Anti-CD79b antibodies and methods of use

ABSTRACT

The invention provides anti-CD79b antibodies and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/960,015 filed on Dec. 4, 2015 which claims the benefit of priority toU.S. Provisional Application No. 62/088,487 filed Dec. 5, 2014, which isherein incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 19, 2018, isnamed P32464-US-2_SL.txt and is 41,735 bytes in size.

FIELD OF THE INVENTION

The present invention relates to anti-CD79b antibodies includinganti-CD79b antibodies comprising a CD3 binding domain (e.g.,anti-CD79b/CD3 T cell dependent bispecific (TDB) antibody) and methodsof using the same.

BACKGROUND

Cell proliferative disorders, such as cancer, are characterized by theuncontrolled growth of cell subpopulations. They are the leading causeof death in the developed world and the second leading cause of death indeveloping countries, with over 12 million new cancer cases diagnosedand 7 million cancer deaths occurring each year. The National CancerInstitute estimates that greater than half a million Americans will dieof cancer in 2013, accounting for nearly one out of every four deaths inthe country. As the elderly population has grown, the incidence ofcancer has concurrently risen, as the probability of developing canceris more than two-fold higher after the age of seventy. Cancer care thusrepresents a significant and ever-increasing societal burden.

CD79b is the signaling component of the B-cell receptor and acts as acovalent heterodimer containing CD79a (i.e., Igα or mb-1) and CD79b(i.e., Igβ or B29). CD79b contains an extracellular immunoglobulin (Ig)domain, a transmembrane domain, and an intracellular signaling domain,an immunoreceptor tyrosine-based activation motif (ITAM) domain. Byusing flow cytometry, surface expression of CD79b has been detected inalmost all non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia(CLL) patients. Dornan et al., Blood 114(13):2721-9 (2009). In additionto its signaling functions, when the B-cell receptor is cross-linked, itis targeted to the major histocompatibility complex class IIcompartment, a lysosome-like compartment, as part of class II antigenpresentation by B cells.

This feature of CD79b biology makes it a particularly attractive targetfor the use of ADCs because antibodies against CD79b are internalizedand delivered to these lysosomal compartments, which are known tocontain protease that can release the cytotoxic drug. Therefore,antibody-drug conjugates (ADC) have been generated (such as thehumanized anti-CD79b antibody (humanized SN8) conjugated tomonomethylauristatin E (MMAE) by a protease cleavable linker), which hasshown to be clinical efficacious for the treatment of NHL. See e.g.,U.S. Pat. No. 8,088,378 and Morschhauser et al., “4457 Updated Resultsof a Phase II Randomized Study (ROMULUS) of Polatuzumab Vedotin orPinatuzumab Vedotin Plus Rituximab in Patients with Relapsed/RefractoryNon-Hodgkin Lymphoma” 56^(th) ASH Annual Meeting and Exposition: Dec.6-9, 2014. Despite the advances in NHL and CLL treatment usinganti-CD79b ADC therapeutics, there still remains an unmet need forimproved therapies for NHL and CLL patients, in particular thoseresistant to anti-CD79b ADC therapies.

Recently, bispecific antibody-based immunotherapies have been developed,which are capable of simultaneously binding cell surface antigens oncytotoxic cells and tumor cells with the intent that the bound cytotoxiccell will destroy the bound tumor cell. Such bispecific antibodies mayhave advantages, e.g., efficacy and/or safety compared to theantibody-drug conjugate. Thus, there is an unmet need in the field forthe development of effective bispecific antibodies for use in cancertreatment.

SUMMARY

The invention provides anti-CD79b antibodies and methods of using thesame. In particular provided herein are anti-CD79b antibodies comprisinga CD79b binding domain and a CD3 binding domain.

In one aspect, provided herein are isolated anti-CD79b antibodies, theantibody comprises a CD79b binding domain comprising the following sixhypervariable regions (HVRs): (a) an HVR-H1 comprising the amino acidsequence of SEQ ID NO: 5; (b) an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 8; (c) an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 9; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO: 10; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 11; and (f) an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 12.

In some embodiments, the CD79b binding domain comprises the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 6; (c)an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In someembodiments, the anti-CD79b antibody comprises (a) a VH sequence havingat least 95% sequence identity to the amino acid sequence of SEQ IDNO:17, 21, 23, 25, 27 or 29; (b) a VL sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO:18, 22, 24,26, 28, or 30; or (c) a VH sequence as in (a) and a VL sequence as in(b). In some embodiments, the anti-CD79b comprises a VH sequence of SEQID NO: 17, 21, 23, 25, 27 or 29. In some embodiments, the anti-CD79bantibody comprises a VL sequence of SEQ ID NO: 18, 22, 24, 26, 28, or30.

In some embodiments, the CD79b binding domain comprises the followingsix HVRs: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c)an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 9; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO: 10; (e) anHVR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and (f) anHVR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In someembodiments, the anti-CD79b antibody comprises (a) a VH sequence havingat least 95% sequence identity to the amino acid sequence of SEQ IDNO:19; (b) a VL sequence having at least 95% sequence identity to theamino acid sequence of SEQ ID NO:20; or (c) a VH sequence as in (a) anda VL sequence as in (b). In some embodiments, the anti-CD79b antibodycomprises a VH sequence of SEQ ID NO:19. In some embodiments, theanti-CD79b antibody comprises a VL sequence of SEQ ID NO:20.

In some embodiments of any of the anti-CD79b antibodies, the CD79bbinding domain binds to SEQ ID NO:63.

In some embodiments of any of the anti-CD79b antibodies, the anti-CD79bantibody (e.g., CD79b binding domain) binds human CD79b with a Kd ofless than about 25 nM as a dual arm, bivalent IgG antibody, e.g., lessthan about any of 10 nM or 5 nM. In some embodiments, Kd is determinedby BIACORE. In some embodiments, Kd is determined by CD79b immobilizedat a low density. In some embodiments of any of the anti-CD79bantibodies, the anti-CD79b antibody (e.g., CD79b binding domain) binds aB cell (e.g., BJAB cell) at an EC₅₀ of less than about 150 ng/mL as adual arm, bivalent IgG antibody, e.g., less than about any of 100 ng/mL,75 ng/mL, or 50 ng/mL. In some embodiments, binding to a B cell isdetermined by FACS. In some embodiments of any of the anti-CD79bantibodies, the anti-CD79b antibody (e.g., CD79b binding domain) bindshuman CD79b binds a B cell (e.g., BJAB cell) at an EC₅₀ of less thanabout 1.5 ug/mL in a monovalent format (e.g., an anti-CD79b bispecificantibody comprising a CD79b and CD3 binding domain), e.g., less thanabout 1 ug/mL, 0.75 ug/mL, 0.5 ug/mL or 0.25 ug/mL. In some embodiments,binding to a B cell is determined by FACS.

In some embodiments of any of the anti-CD79b antibodies, the anti-CD79bantibody is a monoclonal, human, humanized, or chimeric antibody. Insome embodiments of any of the anti-CD79b antibodies, the antibody is anIgG antibody. In some embodiments of any of the anti-CD79b antibodies,the antibody is an antibody fragment that binds CD79b. In someembodiments, the antibody fragment is a Fab, Fab′-SH, Fv, scFv, and/or(Fab′)₂ fragment. In some embodiments of any of the anti-CD79bantibodies, the antibody is a full-length antibody.

In some embodiments of any of the anti-CD79b antibodies, the anti-CD79bantibody comprises an aglycosylation site mutation. In some embodiments,the aglycosylation site mutation is a substitution mutation.

In some embodiments of any of the anti-CD79b antibodies, the anti-CD79bantibody comprises reduced effector function. In some embodiments, theantibody comprises a substitution mutation is at amino acid residueN297, L234, L235, and/or D265 according to EU numbering. In someembodiments, the substitution mutation is selected from the groupconsisting of N297G, N297A, L234A, L235A, and D265A according to EUnumbering. In some embodiments, the antibody comprises an N297Gsubstitution mutation at amino acid residue 297 according to EUnumbering.

In some embodiments of any of the anti-CD79b antibodies, the anti-CD79bantibody is a monospecific antibody (e.g., bivalent, dual arm antibody).

In some embodiments of any of the anti-CD79b antibodies, the anti-CD79bantibody is a multispecific antibody.

In some embodiments of any of the multispecific antibodies, themultispecific antibody comprises a CD3 binding domain. In someembodiments, the CD3 binding domain binds to a human CD3 polypeptide ora cynomolgus monkey (cyno) CD3 polypeptide. In some embodiments, thehuman CD3 polypeptide or the cyno CD3 polypeptide is a human CD3Epolypeptide or a cyno CD3E polypeptide, respectively. In someembodiments, the human CD3 polypeptide or the cyno CD3 polypeptide is ahuman CD3γ polypeptide or a cyno CD3γ polypeptide, respectively.

In some embodiments of any of the multispecific antibodies, the CD3binding domain binds the human CD3E polypeptide with a Kd of 250 nM orlower. In some embodiments, the CD3 binding domain binds the human CD3Epolypeptide with a Kd of 100 nM or lower. In some embodiments, the CD3binding domain binds the human CD3E polypeptide with a Kd of 15 nM orlower. In some embodiments, the CD3 binding domain binds the human CD3Epolypeptide with a Kd of 10 nM or lower. In some embodiments, the CD3binding domain binds the human CD3E polypeptide with a Kd of 5 nM orlower.

In some embodiments of any of the multispecific anti-CD79b antibodies,the CD3 binding domain comprises the following six HVRs: (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO:45; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:46; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:47; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:48; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:49; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:50. In some embodimentsof any of the multispecific anti-CD79b antibodies, the CD3 bindingdomain comprises (a) a VH sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO:59; (b) a VL sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:60;or (c) a VH sequence as in (a) and a VL sequence as in (b). In someembodiments, the CD3 binding domain comprises a VH sequence of SEQ IDNO:59. In some embodiments, the CD3 binding domain comprises a VLsequence of SEQ ID NO:60.

In some embodiments of any of the multispecific anti-CD79b antibodies,the CD3 binding domain comprises the following six HVRs: (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO:39; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:40; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:41; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:42; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:43; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:44. In some embodimentsof any of the multispecific anti-CD79b antibodies, the CD3 bindingdomain comprises (a) a VH sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO:57; (b) a VL sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:58;or (c) a VH sequence as in (a) and a VL sequence as in (b). In someembodiments, the CD3 binding domain comprises a VH sequence of SEQ IDNO:57. In some embodiments, the CD3 binding domain comprises a VLsequence of SEQ ID NO:58.

In some embodiments of any of the multispecific anti-CD79b antibodies,the CD3 binding domain comprises the following six HVRs: (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO:51; (b) an HVR-H2comprising the amino acid sequence of SEQ ID NO:52; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:53; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:54; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:55; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:56. In some embodimentsof any of the multispecific anti-CD79b antibodies, the CD3 bindingdomain comprises (a) a VH sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO:61; (b) a VL sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:62;or (c) a VH sequence as in (a) and a VL sequence as in (b). In someembodiments, the CD3 binding domain comprises a VH sequence of SEQ IDNO:61. In some embodiments, the CD3 binding domain comprises a VLsequence of SEQ ID NO:62.

In some embodiments of any of the multispecific anti-CD79b antibodies,the anti-CD79b antibody has a B cell killing EC₅₀ of less than about 100ng/mL, e.g., less than about any of 50, 25, 20, or 15 ng/mL. In someembodiments, the B cell killing is endogenous B cell killing. In someembodiments, the B cell killing is cell line B cell killing, e.g., BJABcell line, WSU-CLCL2 cell line, OCI-Ly-19 cell line. In some embodimentsof any of the multispecific anti-CD79b antibodies, the anti-CD79bantibody has a cytotoxic T cell activation EC₅₀ is less than about anyof 50 ng/mL, e.g., less than about any of 25 ng/mL or 20 ng/mL. In someembodiments, cytotoxic T cell activation is measured by % of CD69+CD25+T cells in CD8+ T cells.

In some embodiments of any of the multispecific anti-CD79b antibodies,the multispecific antibody is a bispecific antibody.

In some embodiments of any of the multispecific anti-CD79b antibodies,(a) the CD3 binding domain comprises a Fc domain, wherein the Fc domaincomprises T366S, L368A, Y407V, and N297G substitution mutationsaccording EU numbering and (b) the CD79b binding domain comprises a Fcdomain, wherein the Fc domain comprises T366W and N297G substitutionmutations according EU numbering. In some embodiments of any of themultispecific anti-CD79b antibodies, (a) the CD79b binding domaincomprises a Fc domain, wherein the Fc domain comprises T366S, L368A,Y407V, and N297G substitution mutations according EU numbering and (b)the CD3 binding domain comprises a Fc domain, wherein the Fc domaincomprises T366W and N297G substitution mutations according EU numbering.

In some embodiments of any of the multispecific anti-CD79b antibodies,the anti-CD79b antibody comprises one or more heavy chain constantdomains, wherein the one or more heavy chain constant domains areselected from a first CH1 (CH1₁) domain, a first CH2 (CH2₁) domain, afirst CH3 (CH3₁) domain, a second CH1 (CH1₂) domain, second CH2 (CH2₂)domain, and a second CH3 (CH3₂) domain. In some embodiments, at leastone of the one or more heavy chain constant domains is paired withanother heavy chain constant domain. In some embodiments, the CH3₁ andCH3₂ domains each comprise a protuberance or cavity, and wherein theprotuberance or cavity in the CH3₁ domain is positionable in the cavityor protuberance, respectively, in the CH3₂ domain. In some embodiments,the CH3₁ and CH3₂ domains meet at an interface between said protuberanceand cavity. In some embodiments, the CH2₁ and CH2₂ domains each comprisea protuberance or cavity, and wherein the protuberance or cavity in theCH2₁ domain is positionable in the cavity or protuberance, respectively,in the CH2₂ domain. In some embodiments, the CH2₁ and CH2₂ domains meetat an interface between said protuberance and cavity.

Provided herein are also isolated nucleic acids encoding an anti-CD79bantibody described herein. Further provided herein are vectorscomprising an isolated nucleic acid encoding an anti-CD79b antibodydescribed herein. Provided herein are host cells comprising a vectorcomprising an isolated nucleic acid encoding an anti-CD79b antibodydescribed herein. In some embodiments, the host cell is a eukaryotichost cell. In some embodiments, the host cell is a mammalian host cell(e.g., CHO). In some embodiments, the host cell is a prokaryotic hostcell. In some embodiments, the prokaryotic host cell is an E. coli hostcell. Provided herein are further methods of producing the anti-CD79bantibody described herein, wherein the method comprising culturing thehost cell described herein in a culture medium.

Further provided herein are immunoconjugates comprising an anti-CD79bantibody of described herein and a cytotoxic agent.

Provided herein are pharmaceutical compositions comprising theanti-CD79b antibody described herein.

Provided herein are anti-CD79b antibodies as described herein for use asa medicament. Provided herein are anti-CD79b antibody described hereinfor use in treating or delaying progression of a B cell proliferativedisorder or an autoimmune disorder in a subject in need thereof.Provided herein are anti-CD79b antibodies as described herein for use inenhancing immune function in a subject having a B cell proliferativedisorder or an autoimmune disorder. In some embodiments, the B cellproliferative disorder is a cancer. In some embodiments, the B cellproliferative disorder is lymphoma, non-Hodgkins lymphoma (NHL),aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL), and/or mantle cell lymphoma. In someembodiments of any of the B cell proliferative disorders, the B cellproliferative disorder is resistant to treatment with an anti-CD79bantibody drug conjugate (e.g., anti-CD79b MMAE antibody drug conjugate).In some embodiments, the autoimmune disorder is selected from the groupconsisting of rheumatoid arthritis, juvenile rheumatoid arthritis,systemic lupus erythematosus (SLE), Wegener's disease, inflammatorybowel disease, idiopathic thrombocytopenic purpura (ITP), thromboticthrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiplesclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myastheniagravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen'ssyndrome, glomerulonephritis, Neuromyelitis Optica (NMO) and IgGneuropathy.

Provided herein are uses of any of the anti-CD79b antibody describedherein in the manufacture of a medicament for treating or delayingprogression of a cell proliferative disorder or an autoimmune disorder.Provided herein are uses of any of the anti-CD79b antibody describedherein in the manufacture of a medicament for enhancing immune functionin a subject having a cell proliferative disorder or an autoimmunedisorder. In some embodiments, the B cell proliferative disorder is acancer. In some embodiments, the B cell proliferative disorder islymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL), and/or mantle cell lymphoma. In some embodiments of anyof the B cell proliferative disorders, the B cell proliferative disorderis resistant to treatment with an anti-CD79b antibody drug conjugate(e.g., anti-CD79b MMAE antibody drug conjugate). In some embodiments,the autoimmune disorder is selected from the group consisting ofrheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupuserythematosus (SLE), Wegener's disease, inflammatory bowel disease,idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenicpurpura (TTP), autoimmune thrombocytopenia, multiple sclerosis,psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis,vasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen's syndrome,glomerulonephritis, Neuromyelitis Optica (NMO) and IgG neuropathy.

Provided herein are methods of treating or delaying the progression of acell proliferative disorder or an autoimmune disorder in a subject inneed thereof, the method comprising administering to the subject ananti-CD79b antibody described herein. Provided herein are methods ofenhancing immune function in a subject having a cell proliferativedisorder or an autoimmune disorder, the method comprising administeringto the subject an effective amount of an anti-CD79b antibody describedherein. In some embodiments, the B cell proliferative disorder is acancer. In some embodiments, the B cell proliferative disorder islymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL), and/or mantle cell lymphoma. In some embodiments of anyof the B cell proliferative disorders, the B cell proliferative disorderis resistant to treatment with an anti-CD79b antibody drug conjugate(e.g., anti-CD79b MMAE antibody drug conjugate). In some embodiments,the autoimmune disorder is selected from the group consisting ofrheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupuserythematosus (SLE), Wegener's disease, inflammatory bowel disease,idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenicpurpura (TTP), autoimmune thrombocytopenia, multiple sclerosis,psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis,vasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen's syndrome,glomerulonephritis, Neuromyelitis Optica (NMO) and IgG neuropathy.

In some embodiments of any of the methods, the anti-CD79b antibody bindsto (a) a CD3 molecule located on an immune effector cell and (b) a CD79bmolecule located on a B cell. In some embodiments, the anti-CD79bantibody activates the immune effector cell following binding to (a) and(b). In some embodiments of any of the methods, the activated immuneeffector cell is capable of exerting a cytotoxic effect and/or anapoptotic effect on the target cell.

In some embodiments of any of the methods, the method further comprisesadministering to the subject a PD-1 axis binding antagonist or anadditional therapeutic agent. In some embodiments, the PD-1 axis bindingantagonist is a PD-1 binding antagonist. In some embodiments, the PD-1axis binding antagonist is a PD-L1 binding antagonist. In someembodiments, the PD-1 axis binding antagonist is a PD-L2 bindingantagonist.

In some embodiments of any of the methods, the method further comprisesadministering to the subject a glucocorticoid. In some embodiments, theglucocorticoid is dexamethasone.

In some embodiments of any of the methods, the method further comprisesadministering to the subject rituximab.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C shows endogenous B cell killing with anti-CD79b/CD3 TDB (Tcell dependent bispecific) antibodies produced in either K&H format oras bisfabs (various anti-CD79b clones paired with anti-CD3 cloneUCHT1v9). 200,000 PBMCs were incubated with or without anti-CD79b TDBfor 24 hours. At the end of the incubation, the number of live B cellswas counted by gating on CD19+/PI− population. The percent of B cellkilling was calculated as follows: (live B cell number without TDB—liveB cell number with TDB)/(live B cell number without TDB)*100.

FIG. 2A-D shows B cell killing and T cell activation activity of bisfabanti-CD79b/CD3 (CD79b.A7/UCHT1v9). A and B: 200,000 PBMCs were incubatedwith or without anti-CD79b TDB for 24 h. At the end of the incubation,the number of live B cells was counted by gating on CD19+/PI-population.The percent of B cell killing (A) was calculated as follows: (live Bcell number without TDB—live B cell number with TDB)/(live B cell numberwithout TDB)*100; T cell activation (B) was measured by gating onCD69+/CD25+ cells in CD8+ T cell population. C and D: 20,000 BJAB cellsand 100,000 CD8+ T cells were incubated with or without anti-CD79b TDBfor 24 h. At the end of the incubation, the number of live B cells wascounted by gating on CD19+/PI− population. The percent of B cell killing(C) was calculated as follows: (live B cell number without TDB—live Bcell number with TDB)/(live B cell number without TDB)*100; T cellactivation (D) was measured by gating on CD69+/CD25+ cells in CD8+ Tcell population.

FIG. 3A-C shows monovalent or bivalent binding affinity of variousclones of anti-CD79b measured by FACS. BJAB cells were incubated withanti-CD79b antibody (bivalent, dual arm antibody) or anti-CD79b/CD3 TDBantibody as indicated for 30 minutes on ice. At the end of theincubation, cells were washed with ice cold FACS buffer (1×PBS, 2% BSA,2 mM EDTA), followed by incubation with PE-labeled mouse anti-human IgGantibody (BD bioscience #555787). Flow cytometry analysis was done on aBD LSR analyzer. Antibody binding was expressed as Mean FluorescenceIntensity (MFI) of PE fluorophore. A: The bivalent binding affinity ofanti-CD79b clone 2F2 comparing to monovalent binding affinity ofanti-CD79b clones 2F2, SN8v28, and SN8new (as K&H TDBs); B: bivalentbinding affinity of anti-CD79b clones CD79b.F6 and CD79b.A7 comparing tomonovalent binding of anti-CD79b clones CD79b.F6, CD79b.A7, and SN8v28(as bisfab or K&H TDBs); C: bivalent binding affinity of anti-CD79bclone CD79b.A7.v14 comparing to monovalent binding of anti-CD79b cloneCD79b.A7.v14 (as K&H TDBs).

FIG. 4A-B shows alignment of (A) heavy chain variable region (SEQ ID NOS13, 15, 17, 19, 21, 23, 25, 27 and 29, respectively, in order ofappearance) and (B) light chain variable region (SEQ ID NOS 14, 16, 18,20, 22, 24, 26, 28 and 30, respectively, in order of appearance) ofCD79b antibody variants.

FIG. 5A-B shows B cell killing and T cell activation activity ofanti-CD79b/CD3 TDBs (CD79b.A7.v14 paired with either anti-CD3 clone40G5c or 38E4v1). 200,000 PBMCs were incubated with or withoutanti-CD79b TDB for 48 hours. At the end of the incubation, the number oflive B cells was counted by gating on CD19+/PI− population. The percentof B cell killing (A) was calculated as follows: (live B cell numberwithout TDB—live B cell number with TDB)/(live B cell number withoutTDB)*100; T cell activation (B) was measured by gating on CD69⁺/CD25⁺cells in CD8⁺ T cell population.

FIG. 6A-B shows B cell killing and T cell activation activity ofanti-CD79b/CD3 TDBs (CD79b.A7v14/38E4v1). 20,000 BJAB or WSU-DLCL2 cellsand 100,000 CD8+ T cells were incubated with or without anti-CD79b TDBfor 48 hours. At the end of the incubation, the number of live B cellswas counted by gating on CD19+/PI− population. The percent of B cellkilling (A) was calculated as follows: (live B cell number withoutTDB—live B cell number with TDB)/(live B cell number without TDB)*100; Tcell activation (B) was measured by gating on CD69⁺/CD25⁺ cells in CD8⁺T cell population.

FIG. 7A-C shows B cell killing activity of anti-CD79b/CD3 TDB antibody(A7.v14b/38E4v1). 20,000 B lymphoma cells (as indicated) and 100,000CD8⁺ T cells were incubated with or without anti-CD79b TDB for 48 hours.At the end of the incubation, the number of live B cells was counted bygating on CD19+/PI− population. The percent of B cell killing wascalculated as follows: (live B cell number without TDB—live B cellnumber with TDB)/(live B cell number without TDB)*100. A. shows a doseresponse curve of B cell killing for BJAB, WSU-DLCL2, and OCI-LY-19cells, with HT cells as CD79b negative control; B-C. show B cell killingwith 5000 ng/ml Xanti-CD79 TDB (duplicate, average±STD).

FIG. 8A-B shows B cell killing activity of anti-CD79b/CD3 TDB antibody(CD79b.A7.v14b/38E4v1) in vitro and in vivo. BJAB cell variants(BJAB-CD79b ADC-R T1.1 and BJAB-SN8v28vcE CD79b ADC-R T1.2) were derivedfrom non-responsive BJAB xenograft tumors in anti-CD79b-MC-vc-PAB-MMAEtreated mice. A. shows dose response curve of BJAB cell killing invitro: 20,000 BJAB or BJAB variant cells (as indicated) and 100,000 CD8+T cells were incubated with or without anti-CD79b/CD3 TDB antibody(CD79b.A7.v14b/38E4v1) for 48 hours. At the end of the incubation, thenumber of live B cells was counted by gating on CD19+/PI− population.The percent of B cell killing was calculated as follows: (live B cellnumber without TDB—live B cell number with TDB)/(live B cell numberwithout TDB)*100; B. anti-CD79b/CD3 TDB antibody (CD79b.A7.v14b/38E4v1)prevents BJAB tumor growth in vivo: BJAB cells and PBMCs from healthydonor were mixed and inoculated subcutaneously, and mice were thentreated as indicated. Tumor volumes were measured throughout the studyup to 42 days.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

The term “CD79b”, as used herein, refers to any native CD79b from anyvertebrate source, including mammals such as primates (e.g., humans,cynomologus monkey (cyno)) and rodents (e.g., mice and rats), unlessotherwise indicated. Human CD79b is also referred herein to as “Igfβ,”“B29,” “DNA225786” or “PRO36249.” An exemplary CD79b sequence includingthe signal sequence is shown in SEQ ID NO:1. An exemplary CD79b sequencewithout the signal sequence is shown in SEQ ID NO:2. The term “CD79b”encompasses “full-length,” unprocessed CD79b as well as any form ofCD79b that results from processing in the cell. The term alsoencompasses naturally occurring variants of CD79b, e.g., splicevariants, allelic variants and isoforms. The CD79b polypeptidesdescribed herein may be isolated from a variety of sources, such as fromhuman tissue types or from another source, or prepared by recombinant orsynthetic methods. A “native sequence CD79b polypeptide” comprises apolypeptide having the same amino acid sequence as the correspondingCD79b polypeptide derived from nature. Such native sequence CD79bpolypeptides can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native sequence CD79bpolypeptide” specifically encompasses naturally-occurring truncated orsecreted forms of the specific CD79b polypeptide (e.g., an extracellulardomain sequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants of thepolypeptide.

The term “cluster of differentiation 3” or “CD3,” as used herein, refersto any native CD3 from any vertebrate source, including mammals such asprimates (e.g. humans) and rodents (e.g., mice and rats), unlessotherwise indicated, including, for example, CD3ε, CD3γ, CD3α, and CD3βchains. The term encompasses “full-length,” unprocessed CD3 (e.g.,unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of CD3, including, for example, splice variants orallelic variants. CD3 includes, for example, human CD3ε protein (NCBIRefSeq No. NP_000724), which is 207 amino acids in length, and humanCD3γ protein (NCBI RefSeq No. NP_000064), which is 182 amino acids inlength.

The terms “anti-CD79b antibody” and “an antibody that binds to CD79b”refer to an antibody that is capable of binding CD79b with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting CD79b. In one embodiment, the extent ofbinding of an anti-CD79b antibody to an unrelated, non-CD79b protein isless than about 10% of the binding of the antibody to CD79b as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to CD79b has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less,e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M). In certainembodiments, an anti-CD79b antibody binds to an epitope of CD79b that isconserved among CD79b from different species.

The terms “anti-CD3 antibody” and “an antibody that binds to CD3” referto an antibody that is capable of binding CD3 with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting CD3. In one embodiment, the extent of binding of ananti-CD3 antibody to an unrelated, non-CD3 protein is less than about10% of the binding of the antibody to CD3 as measured, e.g., by aradioimmunoassay (RIA). In certain embodiments, an antibody that bindsto CD3 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less, e.g., from10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M). In certain embodiments,an anti-CD3 antibody binds to an epitope of CD3 that is conserved amongCD3 from different species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); and multispecific antibodies formed from antibodyfragments.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32(L1),50-52 (L2), 91-96 (L3), 26-32(H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

By “binding domain” is meant a part of a compound or a molecule thatspecifically binds to a target epitope, antigen, ligand, or receptor.Binding domains include but are not limited to antibodies (e.g.,monoclonal, polyclonal, recombinant, humanized, and chimericantibodies), antibody fragments or portions thereof (e.g., Fabfragments, Fab′2, scFv antibodies, SMIP, domain antibodies, diabodies,minibodies, scFv-Fc, affibodies, nanobodies, and VH and/or VL domains ofantibodies), receptors, ligands, aptamers, and other molecules having anidentified binding partner.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁶, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-CD79b antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

“Isolated nucleic acid encoding an anti-CD3 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result. Aneffective amount herein may vary according to factors such as thedisease state, age, sex, and weight of the patient, and the ability ofthe antibody to elicit a desired response in the individual. Aneffective amount is also one in which any toxic or detrimental effectsof the treatment are outweighed by the therapeutically beneficialeffects. For prophylactic use, beneficial or desired results includeresults such as eliminating or reducing the risk, lessening theseverity, or delaying the onset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as decreasing one or more symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, enhancing effect of another medicationsuch as via targeting, delaying the progression of the disease, and/orprolonging survival. In the case of cancer or tumor, an effective amountof the drug may have the effect in reducing the number of cancer cells;reducing the tumor size; inhibiting (i.e., slow to some extent ordesirably stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and desirably stop) tumor metastasis;inhibiting to some extent tumor growth; and/or relieving to some extentone or more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective amount of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective amount” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

As used herein, “delaying progression” of a disorder or disease means todefer, hinder, slow, retard, stabilize, and/or postpone development ofthe disease or disorder (e.g., a cell proliferative disorder, e.g.,cancer). This delay can be of varying lengths of time, depending on thehistory of the disease and/or individual being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe disease. For example, a late stage cancer, such as development ofmetastasis, may be delayed.

By “reduce” or “inhibit” is meant the ability to cause an overalldecrease, for example, of 20% or greater, of 50% or greater, or of 75%,85%, 90%, 95%, or greater. In certain embodiments, reduce or inhibit canrefer to the effector function of an antibody that is mediated by theantibody Fc region, such effector functions specifically includingcomplement-dependent cytotoxicity (CDC). antibody-dependent cellularcytotoxicity (ADCC), and antibody-dependent cellular phagocytosis(ADCP).

A “chemotherapeutic agent” refers to a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens suchas calusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); Ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®,Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib),proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (seedefinition below); tyrosine kinase inhibitors; serine-threonine kinaseinhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferaseinhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above such as CHOP, an abbreviationfor a combined therapy of cyclophosphamide, doxorubicin, vincristine,and prednisolone; and FOLFOX, an abbreviation for a treatment regimenwith oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens with mixed agonist/antagonist profile, including,tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®),idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, andselective estrogen receptor modulators (SERMs) such as SERM3; pureanti-estrogens without agonist properties, such as fulvestrant(FASLODEX®), and EM800 (such agents may block estrogen receptor (ER)dimerization, inhibit DNA binding, increase ER turnover, and/or suppressER levels); aromatase inhibitors, including steroidal aromataseinhibitors such as formestane and exemestane (AROMASIN®), andnonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®),letrozole (FEMARA®) and aminoglutethimide, and other aromataseinhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®),fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormoneagonists, including leuprolide (LUPRON® and ELIGARD®), goserelin,buserelin, and tripterelin; sex steroids, including progestines such asmegestrol acetate and medroxyprogesterone acetate, estrogens such asdiethylstilbestrol and premarin, and androgens/retinoids such asfluoxymesterone, all transretionic acid and fenretinide; onapristone;anti-progesterones; estrogen receptor down-regulators (ERDs);anti-androgens such as flutamide, nilutamide and bicalutamide; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above.

The term “immunosuppressive agent” as used herein for adjunct therapyrefers to substances that act to suppress or mask the immune system ofthe mammal being treated herein. This would include substances thatsuppress cytokine production, down-regulate or suppress self-antigenexpression, or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077);non-steroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus,glucocorticoids such as cortisol or aldosterone, anti-inflammatoryagents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor,or a leukotriene receptor antagonist; purine antagonists such asazathioprine or mycophenolate mofetil (MMF); alkylating agents such ascyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MHC antigens and MHC fragments;cyclosporin A; steroids such as corticosteroids or glucocorticosteroidsor glucocorticoid analogs, e.g., prednisone, methylprednisolone,including SOLU-MEDROL® methylprednisolone sodium succinate, anddexamethasone; dihydrofolate reductase inhibitors such as methotrexate(oral or subcutaneous); anti-malarial agents such as chloroquine andhydroxychloroquine; sulfasalazine; leflunomide; cytokine or cytokinereceptor antibodies including anti-interferon-alpha, -beta, or -gammaantibodies, anti-tumor necrosis factor(TNF)-alpha antibodies (infliximab(REMICADE®) or adalimumab), anti-TNF-alpha immunoadhesin (etanercept),anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies andanti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptorantibodies and antagonists (such as ACTEMRA™ (tocilizumab)); anti-LFA-1antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies,preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptidecontaining a LFA-3 binding domain (WO 90/08187 published Jul. 26, 1990);streptokinase; transforming growth factor-beta (TGF-beta);streptodornase; RNA or DNA from the host; FK506; RS-61443; chlorambucil;deoxyspergualin; rapamycin; T-cell receptor (Cohen et al., U.S. Pat. No.5,114,721); T-cell receptor fragments (Offner et al., Science, 251:430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO91/01133); BAFF antagonists such as BAFF antibodies and BR3 antibodiesand zTNF4 antagonists (for review, see Mackay and Mackay, TrendsImmunol., 23:113-5 (2002) and see also definition below); biologicagents that interfere with T cell helper signals, such as anti-CD40receptor or anti-CD40 ligand (CD154), including blocking antibodies toCD40-CD40 ligand (e.g., Durie et al., Science, 261: 1328-30 (1993);Mohan et al., J. Immunol., 154: 1470-80 (1995)) and CTLA4-Ig (Finck etal., Science, 265: 1225-7 (1994)); and T-cell receptor antibodies (EP340,109) such as T10B9. Some preferred immunosuppressive agents hereininclude cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF,or methotrexate.

The term “PD-1 axis binding antagonist” refers to a molecule thatinhibits the interaction of a PD-1 axis binding partner with either oneor more of its binding partner, so as to remove T-cell dysfunctionresulting from signaling on the PD-1 signaling axis—with a result beingto restore or enhance T-cell function (e.g., proliferation, cytokineproduction, target cell killing). As used herein, a PD-1 axis bindingantagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist.

The term “PD-1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to one ormore of its binding partners. In a specific aspect, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. Forexample, PD-1 binding antagonists include anti-PD-1 antibodies, antigenbinding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1binding antagonist reduces the negative co-stimulatory signal mediatedby or through cell surface proteins expressed on T lymphocytes mediatedsignaling through PD-1 so as render a dysfunctional T-cell lessdysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist isMDX-1106 (nivolumab) described herein. In another specific aspect, aPD-1 binding antagonist is MK-3475 (lambrolizumab) described herein. Inanother specific aspect, a PD-1 binding antagonist is CT-011(pidilizumab) described herein. In another specific aspect, a PD-1binding antagonist is AMP-224 described herein.

The term “PD-L1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1, B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as torender a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In a specific aspect, ananti-PD-L1 antibody is YW243.55.870 described herein. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. Instill another specific aspect, an anti-PD-L1 antibody is MPDL3280Adescribed herein. In still another specific aspect, an anti-PD-L1antibody is MEDI4736 described herein.

The term “PD-L2 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to one ormore of its binding partners. In a specific aspect, the PD-L2 bindingantagonist inhibits binding of PD-L2 to PD-1. In some embodiments, thePD-L2 antagonists include anti-PD-L2 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L2 witheither one or more of its binding partners, such as PD-1. In oneembodiment, a PD-L2 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L2 so as rendera dysfunctional T-cell less dysfunctional (e.g., enhancing effectorresponses to antigen recognition). In some embodiments, a PD-L2 bindingantagonist is an immunoadhesin.

The term “tumor” refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. The terms “cancer,” “cancerous,” “cell proliferativedisorder,” “proliferative disorder” and “tumor” are not mutuallyexclusive as referred to herein.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia.

The terms “B cell proliferative disorder” refer to disorders that areassociated with some degree of abnormal B cell proliferation. In oneembodiment, the B cell proliferative disorder is cancer.

“B-cell proliferative disorder” include Hodgkin's disease includinglymphocyte predominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma(NHL); follicular center cell (FCC) lymphomas; acute lymphocyticleukemia (ALL); chronic lymphocytic leukemia (CLL); and Hairy cellleukemia. The non-Hodgkins lymphoma include low grade/follicularnon-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediategrade/follicular NHL, intermediate grade diffuse NHL, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, plasmacytoid lymphocyticlymphoma, mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom'smacroglobulinemia. Treatment of relapses of these cancers are alsocontemplated. LPHD is a type of Hodgkin's disease that tends to relapsefrequently despite radiation or chemotherapy treatment. CLL is one offour major types of leukemia. A cancer of mature B cells calledlymphocytes, CLL is manifested by progressive accumulation of cells inblood, bone marrow and lymphatic tissues. Indolent lymphoma is aslow-growing, incurable disease in which the average patient survivesbetween six and 10 years following numerous periods of remission andrelapse.

The term “non-Hodgkin's lymphoma” or “NHL”, as used herein, refers to acancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin'slymphomas can generally be distinguished from non-Hodgkin's lymphomas bythe presence of Reed-Sternberg cells in Hodgkin's lymphomas and theabsence of said cells in non-Hodgkin's lymphomas. Examples ofnon-Hodgkin's lymphomas encompassed by the term as used herein includeany that would be identified as such by one skilled in the art (e.g., anoncologist or pathologist) in accordance with classification schemesknown in the art, such as the Revised European-American Lymphoma (REAL)scheme as described in Color Atlas of Clinical Hematology, ThirdEdition; A. Victor Hoffbrand and John E. Pettit (eds.) (HarcourtPublishers Limited 2000) (see, in particular FIG. 11.57, 11.58 and/or11.59). More specific examples include, but are not limited to, relapsedor refractory NHL, front line low grade NHL, Stage III/IV NHL,chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/orlymphoma, small lymphocytic lymphoma, B cell chronic lymphacyticleukemia and/or prolymphocytic leukemia and/or small lymphocyticlymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/orlymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenicmarginal zone lymphoma, extranodal marginal zone—MALT lymphoma, nodalmarginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacell myeloma, low grade/follicular lymphoma, intermediategrade/follicular NHL, mantle cell lymphoma, follicle center lymphoma(follicular), intermediate grade diffuse NHL, diffuse large B-celllymphoma, aggressive NHL (including aggressive front-line NHL andaggressive relapsed NHL), NHL relapsing after or refractory toautologous stem cell transplantation, primary mediastinal large B-celllymphoma, primary effusion lymphoma, high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, Burkitt's lymphoma, precursor (peripheral) T-celllymphoblastic leukemia and/or lymphoma, adult T-cell lymphoma and/orleukemia, T cell chronic lymphocytic leukemia and/or prolymphacyticleukemia, large granular lymphocytic leukemia, mycosis fungoides and/orSezary syndrome, extranodal natural killer/T-cell (nasal type) lymphoma,enteropathy type T-cell lymphoma, hepatosplenic T-cell lymphoma,subcutaneous panniculitis like T-cell lymphoma, skin (cutaneous)lymphomas, anaplastic large cell lymphoma, angiocentric lymphoma,intestinal T cell lymphoma, peripheral T-cell (not otherwise specified)lymphoma and angioimmunoblastic T-cell lymphoma.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

It is understood that aspects and variations of the invention describedherein include “consisting of” and/or “consisting essentially of”aspects and variations.

II. Compositions and Methods

In one aspect, the invention is based, in part, onanti-CD79b antibodies.In certain embodiments, the anti-CD79b antibodies comprising a CD79bbinding domain and a CD3 binding domain are provided. In certainembodiments, the anti-CD79b antibodies are anti-CD79b T cell dependentbispecific (TDB) antibodies. Antibodies of the invention are useful,e.g., for the diagnosis or treatment of B cell proliferative diseases.

A. Exemplary Anti-CD79b Antibodies

In one aspect, the invention provides isolated antibodies that bind toCD79b. In some embodiments of any of the anti-CD79b antibodies, theCD79b binding domain binds to SEQ ID NO:63.

In some embodiments of any of the anti-CD79b antibodies, the anti-CD79bantibody (e.g., CD79b binding domain) binds human CD79b with a Kd ofless than about 25 nM as a dual arm, bivalent IgG antibody. In someembodiments of any of the anti-CD79b antibodies, the anti-CD79b antibody(e.g., CD79b binding domain) binds human CD79b with a Kd of less thanabout 10 nM. In some embodiments of any of the anti-CD79b antibodies,the anti-CD79b antibody (e.g., CD79b binding domain) binds human CD79bwith a Kd of less than about 5 nM. In some embodiments, the Kd isdetermined by any method described herein, in particular the examples.In some embodiments, Kd is determined by BIACORE. In some embodiments,Kd is determined by CD79b immobilized at a low density.

In some embodiments of any of the anti-CD79b antibodies, the anti-CD79bantibody (e.g., CD79b binding domain) binds a B cell (e.g., BJAB cell)at an EC₅₀ of less than about 150 ng/mL as a dual arm, bivalent IgGantibody. In some embodiments, the EC₅₀ is less than about 100 ng/mL. Insome embodiments, the EC₅₀ is less than about 75 ng/mL. In someembodiments, the EC₅₀ is less than about 50 ng/mL. In some embodiments,the EC₅₀ is determined by any method described herein, in particular theexamples. In some embodiments, the EC₅₀ is the average about any of 5 or10 experiments. In some embodiments, binding to a B cell is determinedby FACS.

In some embodiments of any of the anti-CD79b antibodies, the anti-CD79bantibody (e.g., CD79b binding domain) binds human CD79b binds a B cell(e.g., BJAB cell) at an EC₅₀ of less than about 1.5 ug/mL in amonovalent format (e.g., an anti-CD79b bispecific antibody comprising aCD79b and CD3 binding domain. In some embodiments, the EC₅₀ is less thanabout 1 ug/mL. In some embodiments, the EC₅₀ is less than about 0.75ug/mL. In some embodiments, the EC₅₀ is less than about 0.5 ug/mL. Insome embodiments, the EC₅₀ is less than about 0.25 ug/mL. In someembodiments, the EC₅₀ is determined by any method described herein, inparticular the examples. In some embodiments, the EC₅₀ is the averageabout any of 5 or 10 experiments. In some embodiments, binding to a Bcell is determined by FACS.

Antibody CD79.A7 and Variants Thereof

In one aspect, the invention provides an anti-CD79b antibody comprisinga CD79b binding domain comprising at least one, two, three, four, five,or six HVRs selected from (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:5; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:8; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:9; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:10; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:11; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:12. In some embodiments,HVR-H1 comprises the amino acid sequence of SEQ ID NO:3. In someembodiments, HVR-H1 comprises the amino acid sequence of SEQ ID NO:4. Insome embodiments, HVR-H2 comprises the amino acid sequence of SEQ IDNO:6. In some embodiments, HVR-H2 comprises the amino acid sequence ofSEQ ID NO:7.

In one aspect, the invention provides an anti-CD79b antibody comprisinga CD79b binding domain comprising at least one, at least two, or allthree VH HVR sequences selected from (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:5; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:8; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:9. In one embodiment, the antibody comprisesHVR-H3 comprising the amino acid sequence of SEQ ID NO:9. In anotherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO:9 and HVR-L3 comprising the amino acid sequence ofSEQ ID NO:12. In a further embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:9, HVR-L3 comprising theamino acid sequence of SEQ ID NO:12, and HVR-H2 comprising the aminoacid sequence of SEQ ID NO:8. In a further embodiment, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:5;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:8; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:9. In someembodiments, HVR-H1 comprises the amino acid sequence of SEQ ID NO:3. Insome embodiments, HVR-H1 comprises the amino acid sequence of SEQ IDNO:4. In some embodiments, HVR-H2 comprises the amino acid sequence ofSEQ ID NO:6. In some embodiments, HVR-H2 comprises the amino acidsequence of SEQ ID NO:7. In a further embodiment, the antibody comprises(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:9. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:7; and (c) HVR-H3 comprising the amino acid sequence of SEQID NO:9.

In another aspect, the invention provides an anti-CD79b antibodycomprising a CD79b binding domain comprising at least one, at least two,or all three VL HVR sequences selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:10; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:11; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:12. In one embodiment, the antibody comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:11; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:12.

In another aspect, an anti-CD79b antibody of the invention comprisesCD79b binding domain comprising at (a) a VH domain comprising at leastone, at least two, or all three VH HVR sequences selected from (i)HVR-H1 comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:8, and (iii) HVR-H3comprising an amino acid sequence selected from SEQ ID NO:9; and (b) aVL domain comprising at least one, at least two, or all three VL HVRsequences selected from (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:10, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO:11, and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:12. In some embodiments, HVR-H1 comprises the amino acid sequence ofSEQ ID NO:3. In some embodiments, HVR-H1 comprises the amino acidsequence of SEQ ID NO:4. In some embodiments, HVR-H2 comprises the aminoacid sequence of SEQ ID NO:6. In some embodiments, HVR-H2 comprises theamino acid sequence of SEQ ID NO:7.

In another aspect, the invention provides an anti-CD79b antibodycomprising a CD79b binding domain comprising (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:5; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:8; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO:9; (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO:10; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:11;and (f) HVR-L3 comprising an amino acid sequence selected from SEQ IDNO:12. In another aspect, the invention provides an anti-CD79b antibodycomprising a CD79b binding domain comprising (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:3; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:6; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO:9; (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO:10; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:11;and (f) HVR-L3 comprising an amino acid sequence selected from SEQ IDNO:12. In another aspect, the invention provides an anti-CD79b antibodycomprising a CD79b binding domain comprising (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:7; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO:9; (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO:10; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:11;and (f) HVR-L3 comprising an amino acid sequence selected from SEQ IDNO:12.

In any of the above embodiments, an anti-CD79b antibody is humanized. Inone embodiment, an anti-CD79b antibody comprises HVRs as in any of theabove embodiments, and further comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-CD79b antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:15, 17, 19, 21, 23, 25, 27, and/or 29. In certainembodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CD79b antibody comprising that sequenceretains the ability to bind to CD79b. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO:15, 17, 19, 21, 23, 25, 27, and/or 29. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-CD79b antibody comprisesthe VH sequence in SEQ ID NO:15, 17, 19, 21, 23, 25, 27, and/or 29,including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:5, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:9.

In another aspect, an anti-CD79b antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:16, 18, 20, 22, 24, 26,28, and/or 30. In certain embodiments, a VL sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-CD79B antibodycomprising that sequence retains the ability to bind to CD79b. Incertain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:16, 18, 20, 22, 24,26, 28, and/or 30. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-CD79b antibody comprises the VL sequence inSEQ ID NO:16, 18, 20, 22, 24, 26, 28, and/or 30, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:10; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:11; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:12.

In another aspect, an anti-CD79b antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO:15 and SEQ IDNO:16, respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:17 and SEQ ID NO:18, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:19 and SEQID NO:20, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:21 and SEQ ID NO:22, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:23 and SEQID NO:24, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:25 and SEQ ID NO:26, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO:27 and SEQID NO:28, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO:29 and SEQ ID NO:30, respectively, includingpost-translational modifications of those sequences.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-CD79b antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an anti-CD79b antibody comprising a VH sequence of SEQ IDNO:19 and a VL sequence of SEQ ID NO:20. In certain embodiments, anantibody is provided that binds to an epitope within a fragment of CD79bconsisting of amino acids of SEQ ID NO:63.

In a further aspect of the invention, an anti-CD79b antibody accordingto any of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-CD79bantibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1 antibody or other antibody classor isotype as defined herein.

Antibody SN8.New and Variants Thereof

In one aspect, the invention provides an anti-CD79b antibody comprisinga CD79b binding domain comprising at least one, two, three, four, five,or six HVRs selected from (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:31; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO:32; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33;(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:34; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO:35; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO:36.

In one aspect, the invention provides an anti-CD79b antibody comprisinga CD79b binding domain comprising at least one, at least two, or allthree VH HVR sequences selected from (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:31; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:32; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:33. In one embodiment, the antibody comprisesHVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In anotherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO:33 and HVR-L3 comprising the amino acid sequenceof SEQ ID NO:36. In a further embodiment, the antibody comprises HVR-H3comprising the amino acid sequence of SEQ ID NO:33, HVR-L3 comprisingthe amino acid sequence of SEQ ID NO:36, and HVR-H2 comprising the aminoacid sequence of SEQ ID NO:32. In a further embodiment, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:31;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:32; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:33.

In another aspect, the invention provides an anti-CD79b antibodycomprising a CD79b binding domain comprising at least one, at least two,or all three VL HVR sequences selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:34; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:35; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:36. In one embodiment, the antibody comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO:34; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:35; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO:36.

In another aspect, an anti-CD79b antibody of the invention comprisesCD79b binding domain comprising at (a) a VH domain comprising at leastone, at least two, or all three VH HVR sequences selected from (i)HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:32, and (iii) HVR-H3comprising an amino acid sequence selected from SEQ ID NO:33; and (b) aVL domain comprising at least one, at least two, or all three VL HVRsequences selected from (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:34, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO:35, and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:36.

In another aspect, the invention provides an anti-CD79b antibodycomprising a CD79b binding domain comprising (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:31; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:32; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:34; (e) HVR-L2 comprising the amino acid sequence of SEQ IDNO:35; and (f) HVR-L3 comprising an amino acid sequence selected fromSEQ ID NO:36.

In any of the above embodiments, an anti-CD79b antibody is humanized. Inone embodiment, an anti-CD79b antibody comprises HVRs as in any of theabove embodiments, and further comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework.

In another aspect, an anti-CD79b antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO:37. In certain embodiments, a VH sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-CD79bantibody comprising that sequence retains the ability to bind to CD79b.In certain embodiments, a total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:37. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti-CD79b antibodycomprises the VH sequence in SEQ ID NO:37, including post-translationalmodifications of that sequence. In a particular embodiment, the VHcomprises one, two or three HVRs selected from: (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:31, (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:32, and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:33.

In another aspect, an anti-CD79b antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:38. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-CD79B antibody comprising that sequenceretains the ability to bind to CD79b. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO:38. In certain embodiments, the substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Optionally, the anti-CD79b antibody comprises the VL sequence in SEQ IDNO:38, including post-translational modifications of that sequence. In aparticular embodiment, the VL comprises one, two or three HVRs selectedfrom (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:34; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO:35; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO:36.

In another aspect, an anti-CD79b antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO:37 and SEQ IDNO:38, respectively, including post-translational modifications of thosesequences.

In a further aspect of the invention, an anti-CD79b antibody accordingto any of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-CD79bantibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1 antibody or other antibody classor isotype as defined herein.

In a further aspect, anti-CD79b antibodies according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g., 10⁻⁸M or less, e.g., from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881(1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CM5 chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (kon) anddissociation rates (koff) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiokoff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). Ifthe on-rate exceeds 106 M-1 s-1 by the surface plasmon resonance assayabove, then the on-rate can be determined by using a fluorescentquenching technique that measures the increase or decrease influorescence emission intensity (excitation=295 nm; emission=340 nm, 16nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) inPBS, pH 7.2, in the presence of increasing concentrations of antigen asmeasured in a spectrometer, such as a stop-flow equipped spectrophometer(Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sri, USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for CD79b and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of CD79b. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express CD79b. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

In one aspect, the invention provides isolated anti-CD79b antibodiesthat bind to CD79b and CD3 (i.e., comprising a CD79b binding domain anda CD3 binding domain). In certain embodiments, one of the bindingspecificities is for CD3 (e.g., CD3ε or CD3γ) and the other is CD79b. Insome embodiments, the CD3 binding domain binds to a human CD3polypeptide or a cynomolgus monkey (cyno) CD3 polypeptide. In someembodiments, the human CD3 polypeptide or the cyno CD3 polypeptide is ahuman CD3ε polypeptide or a cyno CD3ε polypeptide, respectively. In someembodiments, the human CD3 polypeptide or the cyno CD3 polypeptide is ahuman CD3γ polypeptide or a cyno CD3γ polypeptide, respectively. Incertain embodiments, an anti-CD79b antibody is provided comprising a CD3binding domain which binds to an epitope within a fragment of CD3 (e.g.,human CD3ε) consisting of amino acids 1-26 or 1-27 of human CD3ε. Insome embodiments, the anti-CD79b antibody is a bispecific antibody. Insome embodiments, the anti-CD79b antibody is a bispecific IgG antibody.

In some embodiments, CD3 binding domain binds the human CD3ε polypeptidewith a Kd of 250 nM or lower. In some embodiments, the CD3 bindingdomain binds the human CD3ε polypeptide with a Kd of 100 nM or lower. Insome embodiments, the CD3 binding domain binds the human CD3εpolypeptide with a Kd of 15 nM or lower. In some embodiments, CD3binding domain binds the human CD3ε polypeptide with a Kd of 10 nM orlower. In some embodiments, CD3 binding domain binds the human CD3εpolypeptide with a Kd of 5 nM or lower.

In some embodiments of any of the multispecific antibodies, e.g., abispecific antibody, that bind to CD79b and CD3, comprises a CD3 bindingdomain, wherein the CD3 binding domain comprises the hypervariableregions (HVRs) (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:39; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:40;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:41; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:42; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:43; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:44. In some embodiments,the CD3 binding domain comprises (a) a VH domain comprising (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:39, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:40, and (iii) HVR-H3comprising an amino acid sequence selected from SEQ ID NO:41; and (b) aVL domain comprising (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:42, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO:43, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:44. In some instances, the CD3 binding domain may have a heavy chainvariable (VH) domain including an amino acid sequence having at least90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:57and/or a light chain variable (VL) domain comprising an amino acidsequence having at least 90% sequence identity (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO:58. In some instances, the CD3 binding domain mayhave a VH domain comprising the amino acid sequence of SEQ ID NO:57 anda VL domain comprising the amino acid sequence of SEQ ID NO:58. In aparticular instance, the CD3 binding domain can be 40G5c, or aderivative or clonal relative thereof.

For example, in some embodiments, the anti-CD79b antibody comprises (i)a CD79b binding domain comprising the HVRs (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:7; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO:9; (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO:10; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:11;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:12 and(ii) a CD3 binding domain comprising the HVRs (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:39; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:40; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:41; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:42; (e) HVR-L2 comprising the amino acid sequence of SEQ IDNO:43; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO:44. In some embodiments, the anti-CD79b antibody comprises (i) aCD79b binding domain comprising (a) a VH domain comprising the aminoacid sequence of SEQ ID NO:19 and (b) a VL domain comprising the aminoacid sequence of SEQ ID NO:20 and (ii) a CD3 binding domain comprising(a) a VH domain comprising the amino acid sequence of SEQ ID NO:57 and(b) a VL domain comprising the amino acid sequence of SEQ ID NO:58.

In some embodiments of any of the multispecific antibodies, e.g. abispecific antibody, that bind to CD79b and CD3, the CD3 binding domaincomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:45;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:46; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:47; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:48; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:49; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:50. In some embodiments,the CD3 binding domain comprises (a) a VH domain comprising (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:45, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:46, and (iii) HVR-H3comprising an amino acid sequence selected from SEQ ID NO:47; and (b) aVL domain comprising (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:48, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO:49, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:50. In some instances, the CD3 binding domain may have a VH domaincomprising an amino acid sequence having at least 90% sequence identity(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO:59 and/or a VL domaincomprising an amino acid sequence having at least 90% sequence identity(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO:60. In some instances, theCD3 binding domain may have a VH domain comprising the amino acidsequence of SEQ ID NO:59 and a VL domain comprising the amino acidsequence of SEQ ID NO:60. In a particular instance, the CD3 bindingdomain can be 38E4v1, or a derivative or clonal relative thereof.

For example, in some embodiments, the anti-CD79b antibody comprises (i)a CD79b binding domain comprising the HVRs (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:7; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO:9; (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO:10; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:11;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:12 and(ii) a CD3 binding domain comprising the HVRs (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:45; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:46; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:47; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:48; (e) HVR-L2 comprising the amino acid sequence of SEQ IDNO:49; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO:50. In some embodiments, the anti-CD79b antibody comprises (i) aCD79b binding domain comprising (a) a VH domain comprising the aminoacid sequence of SEQ ID NO:19 and (b) a VL domain comprising the aminoacid sequence of SEQ ID NO:20 and (ii) a CD3 binding domain comprising(a) a VH domain comprising the amino acid sequence of SEQ ID NO:59 and(b) a VL domain comprising the amino acid sequence of SEQ ID NO:60.

In some embodiments of any of the multispecific antibodies, e.g. abispecific antibody, that bind to CD79b and CD3, the CD3 binding domaincomprises from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO:51; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:52;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:53; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO:54; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO:55; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:56. In some embodiments,the CD3 binding domain comprises (a) a VH domain comprising (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:51, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:52, and (iii) HVR-H3comprising an amino acid sequence selected from SEQ ID NO:53; and (b) aVL domain comprising (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:54, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO:55, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:56. In some instances, the anti-CD3 antibody may have a VH domaincomprising an amino acid sequence having at least 90% sequence identity(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO:61 and/or a VL domaincomprising an amino acid sequence having at least 90% sequence identity(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO:62. In some instances, theCD3 binding domain may have a VH domain comprising the amino acidsequence of SEQ ID NO:61 and a VL domain comprising the amino acidsequence of SEQ ID NO:62. In a particular instance, the anti-CD3antibody can be UCHT1.v9, or a derivative or clonal relative thereof.

For example, in some embodiments, the anti-CD79b antibody comprises (i)a CD79b binding domain comprising the HVRs (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:7; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO:9; (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO:10; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:11;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:12 and(ii) a CD3 binding domain comprising the HVRs (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:51; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:52; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:53; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:54; (e) HVR-L2 comprising the amino acid sequence of SEQ IDNO:55; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO:56. In some embodiments, the anti-CD79b antibody comprises (i) aCD79b binding domain comprising (a) a VH domain comprising the aminoacid sequence of SEQ ID NO:19 and (b) a VL domain comprising the aminoacid sequence of SEQ ID NO:20 and (ii) a CD3 binding domain comprising(a) a VH domain comprising the amino acid sequence of SEQ ID NO:61 and(b) a VL domain comprising the amino acid sequence of SEQ ID NO:62.

In some embodiments of any of the multispecific anti-CD79b antibodies,the anti-CD79b antibody has a B cell killing EC₅₀ of less than about 100ng/mL. In some embodiments, the EC₅₀ is less than about 50 ng/mL. Insome embodiments, the EC₅₀ is less than about 25 ng/mL. In someembodiments, the EC₅₀ is less than about 20 ng/mL. In some embodiments,the EC₅₀ is less than about 15 ng/mL. In some embodiments, the B cellkilling is endogenous B cell killing. In some embodiments, the B cellkilling is cell line B cell killing, e.g., BJAB cell line, WSU-CLCL2cell line, OCI-Ly-19 cell line. In some embodiments, the EC₅₀ isdetermined by any method described herein, in particular the examples.In some embodiments, the EC₅₀ is the average of about any of 5 or 10experiments. In some embodiments, the EC₅₀ is the average of about anyof 5 or 10 donors.

In some embodiments of any of the multispecific anti-CD79b antibodies,the anti-CD79b antibody kills at least about 60% of B cells at 5000ng/mL. In some embodiments, the anti-CD79b antibody kills at least about80% of B cells at 5000 ng/mL. the anti-CD79b antibody kills at leastabout 90% of B cells at 5000 ng/mL. For example, in some embodiments,the B cells are one or more of the B cell lines SU-CHL-6, CoHH2, BJAB,WSU-DLCL2, Sc-1, SU-CHL-8, GRANTA-519, Nalm-6, Ramos, and/or OCI-Ly-19.In some embodiments, the EC₅₀ is determined by any method describedherein, in particular the examples. In some embodiments, the EC₅₀ is theaverage of about any of 5 or 10 experiments. In some embodiments, theEC₅₀ is the average of about any of 5 or 10 donors.

In some embodiments of any of the multispecific anti-CD79b antibodies,the anti-CD79b antibody has a cytotoxic T cell activation EC₅₀ is lessthan about any of 50 ng/mL. In some embodiments, the anti-CD79b antibodyhas a cytotoxic T cell activation EC₅₀ is less than about 25 ng/mL. Insome embodiments, the anti-CD79b antibody has a cytotoxic T cellactivation EC₅₀ is less than about less than 20 ng/mL. In someembodiments, cytotoxic T cell activation is measured by % of CD69+CD25+T cells in CD8+ T cells. In some embodiments, the EC₅₀ is determined byany method described herein, in particular the examples. In someembodiments, the EC₅₀ is the average of about any of 5 or 10experiments. In some embodiments, the EC₅₀ is the average of about anyof 5 or 10 donors.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see,e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to CD79b as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g., bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32(1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivoclearance/half-life determinations can also be performed using methodsknown in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol.18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

In some aspects the anti-CD79b antibody (e.g., anti-CD79b TDB antibody)comprises an Fc region comprising an N297G mutation.

In some embodiments, the anti-CD79b antibody comprising the N297Gmutation comprises one or more heavy chain constant domains, wherein theone or more heavy chain constant domains are selected from a first CH1(CH1₁) domain, a first CH2 (CH2₁) domain, a first CH3 (CH3₁) domain, asecond CH1 (CH1₂) domain, second CH2 (CH2₂) domain, and a second CH3(CH3₂) domain. In some instances, at least one of the one or more heavychain constant domains is paired with another heavy chain constantdomain. In some instances, the CH3₁ and CH3₂ domains each comprise aprotuberance or cavity, and wherein the protuberance or cavity in theCH3₁ domain is positionable in the cavity or protuberance, respectively,in the CH3₂ domain. In some instances, the CH3₁ and CH3₂ domains meet atan interface between said protuberance and cavity. In some instances,the CH2₁ and CH2₂ domains each comprise a protuberance or cavity, andwherein the protuberance or cavity in the CH2₁ domain is positionable inthe cavity or protuberance, respectively, in the CH2₂ domain. In otherinstances, the CH2₁ and CH2₂ domains meet at an interface between saidprotuberance and cavity. In some instances, the anti-CD3 antibody is anIgG1 antibody.

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-CD79b antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-CD79b antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-CD79b antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-CD79b antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with an anti-CD79b antibody described herein forbinding to CD79b. In certain embodiments, such a competing antibodybinds to the same epitope (e.g., a linear or a conformational epitope)that is bound by an anti-CD79b antibody described herein. Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized CD79b is incubated in asolution comprising a first labeled antibody that binds to CD79b (e.g.,an anti-CD79b antibody described herein) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to CD79b. The second antibody may be present in a hybridomasupernatant. As a control, immobilized CD79b is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to CD79b, excess unbound antibody is removed, and theamount of label associated with immobilized CD79b is measured. If theamount of label associated with immobilized CD79b is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to CD79b. See Harlow and Lane (1988) Antibodies: ALaboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-CD79b antibodies(e.g., anti-CD79b/CD3 TDB antibody) thereof having biological activity.Biological activity may include, e.g., the ability to inhibit cellgrowth or proliferation (e.g., “cell killing” activity), the ability toinduce cell death, including programmed cell death (apoptosis), orantigen binding activity. Antibodies having such biological activity invivo and/or in vitro are also provided.

In some embodiments, the activity comprises ability to support B cellkilling and/or the activation of the cytotoxic T cells. In certainembodiments, an anti-CD79b antibody (e.g., anti-CD79b/CD3 TDB antibody)of the invention is tested for such B cell killing and/or the activationof the cytotoxic effect of T cells biological activity by any of themethods described herein, in particular the Examples. In someembodiments of any of these activity assays, PBMCs may be isolated fromwhole blood of healthy donors by Ficoll separation. In particular, humanblood may be collected in heparinized syringes, and PBMC were isolatedusing Leucosep and Ficoll Paque Plus. If needed CD4+T and CD8+ T cellsmay be separated with Miltenyi kits according to manufacturer'sinstructions.

Further, cells may be washed in RPMI medium containing 10% FBS,supplemented with GlutaMax, penicillin & streptomycin, and ˜0.2 millionsuspended cells were added to a 96-well U-bottom plate. Cells may becultured in RPMI1640 supplemented with 10% FBS at 37° C. in a humidifiedstandard cell culture incubator. For BJAB cell killing assays, 20,000BJAB cells may be incubated with effector cells either as huPBMCs orpurified T cells as indicated ratios per assay, in the presence ofvarious concentrations of TDB antibodies for 24 hours. For endogenous Bcell killing assays, 200,000 huPBMCs may be incubated with variousconcentrations of TDB antibodies for 24 hours.

After culturing, cells may be washed with FACS buffer (0.5% BSA, 0.05%Na Azide in PBS). Cells may then be stained in FACS buffer, washed withFACS buffer and suspend in 100 ul of FACS buffer containing 1 ug/mlPropidium Iodide. Data may be collected on a FACSCalibur flow cytometerand analyzed using FlowJo. Live B cells may be gated out as PI-CD19+ orPI-CD20+ B cells by FACS, and absolute cell count may be obtained withFITC beads added to reaction mix as internal counting control. % of cellkilling may be calculated based on non-TDB treated controls. Activated Tcells may be detected by CD69 and CD25 surface expression usinganti-CD69-FITC and anti-CD25-PE.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CD79bantibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁶, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or 1123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In one aspect, anti-CD79b antibodies of the invention are useful fordetecting the presence of CD79b in a biological sample. The term“detecting” as used herein encompasses quantitative or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue. In certain embodiments, such tissues include normal and/orcancerous tissues that express CD79b at higher levels relative to othertissues, for example, B cells and/or B cell associated tissues.

In one embodiment, an anti-CD79b antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of CD79b in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-CD79b antibody as described herein under conditionspermissive for binding of the anti-CD79b antibody to CD79b, anddetecting whether a complex is formed between the anti-CD79b antibodyand CD79b. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-CD79b antibody is used to select subjects eligiblefor therapy with an anti-CD79b antibody, e.g. where CD79b is a biomarkerfor selection of patients.

Exemplary cell proliferative disorders that may be diagnosed using anantibody of the invention include a B cell disorder and/or a B cellproliferative disorder including, but not limited to, lymphoma,non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL,relapsed indolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

Certain other methods can be used to detect binding of anti-CD79bantibodies to CD79b. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, labeled anti-CD79b antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-CD79b antibody as describedherein are prepared by mixing such antibody having the desired degree ofpurity with one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, in addition to an anti-CD79b antibody, it may bedesirable to include in the one formulation, an additional antibody,e.g., a second anti-CD79b antibody which binds a different epitope onthe CD79b polypeptide, or an antibody to some other target such as agrowth factor that affects the growth of the particular cancer.Alternatively, or additionally, the composition may further comprise achemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitoryagent, anti-hormonal agent, and/or cardioprotectant. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-CD79b antibodies (e.g., anti-CD79b/CD3 TDB antibody)provided herein may be used in therapeutic methods.

In one aspect, an anti-CD79b antibody (e.g., anti-CD79b/CD3 TDBantibody) for use as a medicament is provided. In further aspects, ananti-CD79b antibody (e.g., anti-CD79b/CD3 TDB antibody) for use intreating or delaying progression of a cell proliferative disorder (e.g.,cancer and/or B cell proliferative disease) is provided. In certainembodiments, an anti-CD79b antibody (e.g., anti-CD79b/anti-CD3bispecific antibody) for use in a method of treatment is provided. Incertain embodiments, the invention provides an anti-CD79b antibody(e.g., anti-CD79b/CD3 TDB antibody) for use in a method of treating anindividual having a cell proliferative disorder comprising administeringto the individual an effective amount of the anti-CD79b antibody (e.g.,anti-CD79b/CD3 TDB antibody). In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, for example, as described below.In further embodiments, the invention provides an anti-CD79b antibody(e.g., anti-CD79b/CD3 TDB antibody) for use in enhancing immune functionin an individual having a cell proliferative disorder. In certainembodiments, the invention provides an anti-CD79b antibody (e.g.,anti-CD79b/CD3 TDB antibody) for use in a method of enhancing immunefunction in an individual having a cell proliferative disordercomprising administering to the individual an effective of theanti-CD79b antibody (e.g., anti-CD79b/CD3 TDB antibody) to activateeffector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells), expand(increase) an effector cell population, and/or kill a target cell (e.g.,target B cell). An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides for the use of an anti-CD79bantibody (e.g., anti-CD79b/CD3 TDB antibody)) in the manufacture orpreparation of a medicament. In one embodiment, the medicament is fortreatment of a cell proliferative disorder (e.g., cancer and/or B cellproliferative disorder). In a further embodiment, the medicament is foruse in a method of treating a cell proliferative disorder comprisingadministering to an individual having a cell proliferative disorder aneffective amount of the medicament. In one such embodiment, the methodfurther comprises administering to the individual an effective amount ofat least one additional therapeutic agent, for example, as describedbelow. In a further embodiment, the medicament is for activatingeffector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells),expanding (increasing) an effector cell population, and/or killingtarget cells (e.g., target B cells) in the individual. In a furtherembodiment, the medicament is for use in a method of enhancing immunefunction in an individual having a cell proliferative disorder or anautoimmune disorder comprising administering to the individual an amounteffective of the medicament to activate effector cells (e.g., T cells,e.g., CD8+ and/or CD4+ T cells), expand (increase) an effector cellpopulation, and/or kill a target cell (e.g., target B cell). An“individual” according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating a cellproliferative disorder (e.g., cancer and/or B cell proliferativedisorder). In one embodiment, the method comprises administering to anindividual having such a cell proliferative disorder an effective amountof an anti-CD79b antibody (e.g., anti-CD79b/CD3 TDB antibody). In onesuch embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, for example, as described below. An “individual” according to anyof the above embodiments may be a human.

In a further aspect, the invention provides a method for enhancingimmune function in an individual having a cell proliferative disorder inan individual having a cell proliferative disorder. In one embodiment,the method comprises administering to the individual an effective amountof an anti-CD79b antibody (e.g., anti-CD79b/CD3 TDB antibody) toactivate effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells),expand (increase) an effector cell population, and/or kill a target cell(e.g., target B cell). In one embodiment, an “individual” is a human.

An anti-CD79b antibody (e.g., anti-CD79b/CD3 TDB antibody) of theinvention may be used in, for example, in vitro, ex vivo, and in vivotherapeutic methods. In one aspect, the invention provides methods forinhibiting cell growth or proliferation, either in vivo or in vitro, themethod comprising exposing a cell to an anti-CD79b antibody thereofunder conditions permissive for binding to CD79b. “Inhibiting cellgrowth or proliferation” means decreasing a cell's growth orproliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or 100%, and includes inducing cell death. In certain embodiments,the cell is a tumor cell. In certain embodiments, the cell is a B cell.In certain embodiments, the cell is a xenograft, e.g., as exemplifiedherein.

In one aspect, an anti-CD79b antibody (e.g., anti-CD79b/CD3 TDBantibody) of the invention is used to treat or prevent a B cellproliferative disorder. In certain embodiments, the cell proliferativedisorder is associated with increased expression and/or activity ofCD79b. For example, in certain embodiments, the B cell proliferativedisorder is associated with increased expression of CD79b on the surfaceof a B cell. In certain embodiments, the B cell proliferative disorderis a tumor or a cancer. Examples of B cell proliferative disorders to betreated by the antibodies of the invention include, but are not limitedto, lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL), and mantle cell lymphoma. In some embodiments of any ofthe B cell proliferative disorders, the B cell proliferative disorder isresistant to treatment with an anti-CD79b immunoconjugate (e.g.,anti-CD79b MMAE immunoconjugate).

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-CD79b antibodies (e.g., anti-CD79b/CD3 TDBantibody) provided herein, e.g., for use in any of the above therapeuticmethods. In one embodiment, a pharmaceutical formulation comprises anyof the anti-CD79b antibodies provided herein and a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical formulationcomprises any of the anti-CD79b antibodies (e.g., anti-CD79b/CD3 TDBantibody) provided herein and at least one additional therapeutic agent,e.g., as described below.

In one embodiment, B-cell proliferative disease includes, but is notlimited to, lymphomas (e.g., B-Cell Non-Hodgkin's lymphomas (NHL)) andlymphocytic leukemias. Such lymphomas and lymphocytic leukemias includee.g. a) follicular lymphomas, b) Small Non-Cleaved CellLymphomas/Burkitt's lymphoma (including endemic Burkitt's lymphoma,sporadic Burkitt's lymphoma and Non-Burkitt's lymphoma), c) marginalzone lymphomas (including extranodal marginal zone B-cell lymphoma(Mucosa-associated lymphatic tissue lymphomas, MALT), nodal marginalzone B-cell lymphoma and splenic marginal zone lymphoma), d) Mantle celllymphoma (MCL), e) Large Cell Lymphoma (including B-cell diffuse largecell lymphoma (DLCL), Diffuse Mixed Cell Lymphoma, ImmunoblasticLymphoma, Primary Mediastinal B-Cell Lymphoma, AngiocentricLymphoma-Pulmonary B-Cell Lymphoma), f) hairy cell leukemia, g)lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL)/smalllymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasmacell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma,and/or j) Hodgkin's disease.

In some embodiments of any of the methods, the B-cell proliferativedisorder is cancer. In some embodiments, the B-cell proliferativedisorder is lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), or mantle cell lymphoma. In someembodiments, the B-cell proliferative disorder is NHL, such as indolentNHL and/or aggressive NHL. In some embodiments, the B-cell proliferativedisorder is indolent follicular lymphoma or diffuse large B-celllymphoma. In some embodiments of any of the B cell proliferativedisorders, the B cell proliferative disorder is resistant to treatmentwith an anti-CD79b immunoconjugate (e.g., anti-CD79b MMAEimmunoconjugate).

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent and/or adjuvant. In certain embodiments, an additionaltherapeutic agent is a cytotoxic agent, a chemotherapeutic agent, or agrowth inhibitory agent. In one of such embodiments, a chemotherapeuticagent is an agent or a combination of agents such as, for example,cyclophosphamide, hydroxydaunorubicin, adriamycin, doxorubincin,vincristine (Oncovin™) prednisolone, CHOP, CHP, CVP, or COP, orimmunotherapeutics such as anti-CD20 (e.g., Rituxan®) or anti-VEGF(e.g., Avastin®), wherein the combination therapy is useful in thetreatment of cancers and/or B cell disorders such as B cellproliferative disorders including lymphoma, non-Hodgkins lymphoma (NHL),aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL), and mantle cell lymphoma. In otherembodiments, for instance, an antibody of the invention may beco-administered with at least one additional therapeutic agent. Incertain embodiments, an additional therapeutic agent is achemotherapeutic agent, growth inhibitory agent, cytotoxic agent, agentused in radiation therapy, anti-angiogenesis agent, apoptotic agent,anti-tubulin agent, or other agent, such as a epidermal growth factorreceptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor),HER1/EGFR inhibitor (e.g., erlotinib (Tarceva™), platelet derived growthfactor inhibitor (e.g., Gleevec™ (Imatinib Mesylate)), a COX-2 inhibitor(e.g., celecoxib), interferon, cytokine, antibody other than theanti-CD3 antibody of the invention, such as an antibody that bind to oneor more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS,APRIL, BCMA VEGF, or VEGF receptor(s), TRAIL/Apo2, or another bioactiveor organic chemical agent.

In some embodiments, the methods may further comprise an additionaltherapy. The additional therapy may be radiation therapy, surgery,chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,immunotherapy, bone marrow transplantation, nanotherapy, monoclonalantibody therapy, or a combination of the foregoing. The additionaltherapy may be in the form of adjuvant or neoadjuvant therapy. In someembodiments, the additional therapy is the administration of smallmolecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. In some embodiments, the additional therapy isgamma irradiation. In some embodiments, the additional therapy may be aseparate administration of one or more of the therapeutic agentsdescribed above.

In some embodiments of any of the methods, the additional therapeuticagent is a glucocorticoid. In some embodiments, the glucocorticoid isselected from the group consisting of dexamethasone, hydrocortisone,cortisone, prednisolone, prednisone, methylprednisone, triamcinolone,paramethasone, betamethasone, fludrocortisone, and pharmaceuticallyacceptable esters, salts, and complexes thereof. In some embodiments,the glucocorticoid is dexamethasone. In some embodiments, theglucocorticoid is a pharmaceutically acceptable ester, salt, or complexof dexamethasone. In some embodiments, the glucocorticoid isdexamethasone.

In some embodiments of any of the methods, the additional therapycomprises an anti-CD20 antibody. In some embodiments, the anti-CD20antibody is rituximab. In some embodiments, the anti-CD20 antibody is ahumanized B-Ly1 antibody. In some embodiments, the humanized B-Ly1antibody is obinituzumab. In some embodiments, the anti-CD20 antibody isofatumumab, ublituximab, and/or ibritumomab tiuxetan.

In some embodiments of any of the methods, the additional therapycomprises an alkylating agent. In some embodiments, the alkylating agentis 4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoicacid and salts thereof. In some embodiments, the alkylating agent isbendamustine.

In some embodiments of any of the methods, the additional therapycomprises a BCL-2 inhibitor. In some embodiments, the BCL-2 inhibitor is4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamideand salts thereof. In some embodiments, the BCL-2 inhibitor isvenetoclax (CAS #: 1257044-40-8).

In some embodiments of any of the methods, the additional therapycomprises a phosphoinositide 3-kinase (PI3K) inhibitor. In someembodiments, the PI3K inhibitor inhibits delta isoform PI3K (i.e.,P110δ). In some embodiments, the PI3K inhibitor is5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinoneand salts thereof. In some embodiments, the PI3K inhibitor is idelalisib(CAS #: 870281-82-6). In some embodiments, the PI3K inhibitor inhibitsalpha and delta isoforms of PI3K. In some embodiments, the PI3Kinhibitor is2-{3-[2-(1-Isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamideand salts thereof.

In some embodiments of any of the methods, the additional therapycomprises a Bruton's tyrosine kinase (BTK) inhibitor. In someembodiments, the BTK inhibitor is1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-oneand salts thereof. In some embodiments, the BTK inhibitor is ibrutinib(CAS #: 936563-96-1).

In some embodiments of any of the methods, the additional therapycomprises thalidomide or a derivative thereof. In some embodiments, thethalidomide or a derivative thereof is (RS)-3-(4-Amino-1-oxo1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione and salts thereof. Insome embodiments, the thalidomide or a derivative thereof islendalidomide (CAS #: 191732-72-6).

In some embodiments of any of the methods, the additional therapycomprises one or more of cyclophosphamide, doxorubicin, vincristine, orprednisolone (CHOP). In some embodiments, the additional therapy furthercomprises an anti-CD20 antibody as described above (e.g., GA-101 and/orRituxan).

In some embodiments of any of the methods, the additional therapycomprises one or more of cyclophosphamide, doxorubicin, or prednisolone(CHP). In some embodiments, the additional therapy further comprises ananti-CD20 antibody as described above (e.g., GA-101 and/or Rituxan). Insome embodiments, the additional therapy further comprises an anti-CD79bantibody drug conjugate. In some embodiments, the anti-CD79b antibodydrug conjugate is anti-CD79b-MC-vc-PAB-MMAE. In some embodiments, theanti-CD79b antibody drug conjugate is described in any one of U.S. Pat.No. 8,088,378 and/or US 2014/0030280, which are hereby incorporated byreference in their entirety. In some embodiments, the anti-CD79bantibody drug conjugate is polatuzumab vedotin. In some embodiments ofany of the B cell proliferative disorders, the B cell proliferativedisorder is resistant to treatment with an anti-CD79b antibody drugconjugate (e.g., anti-CD79b MMAE antibody drug conjugate). In someembodiments, the anti-CD79b antibody drug conjugate is polatuzumabvedotin.

In some embodiments of any of the methods, the additional therapycomprises a PD-1 axis binding antagonist. In some embodiments of any ofthe methods, the additional therapy comprises a PD-1 binding antagonist.In some embodiments of any of the methods, the additional therapycomprises a PD-L1 binding antagonist. In some embodiments of any of themethods, the additional therapy comprises a PD-L2 binding antagonist.

In some embodiments of any of the methods, an antibody of the invention(and any additional therapeutic agent) can be administered by anysuitable means, including parenteral, intrapulmonary, and intranasal,and, if desired for local treatment, intralesional administration.Parenteral infusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion are contemplated herein. In some embodiments, theadministration is subcutaneous.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments.

As a general proposition, the therapeutically effective amount of theanti-CD79b antibody (e.g., anti-CD79b/CD3 TDB antibody) administered tohuman will be in the range of about 0.01 to about 100 mg/kg of patientbody weight whether by one or more administrations. In some embodiments,the antibody used is about 0.01 to about 45 mg/kg, about 0.01 to about40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg,about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example.In one embodiment, an anti-CD79b antibody (e.g., anti-CD79b/CD3 TDBantibody) described herein is administered to a human at a dose of about100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of21-day cycles. The dose may be administered as a single dose or asmultiple doses (e.g., 2 or 3 doses), such as infusions. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody wouldbe in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one ormore doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, for example, every week or every threeweeks (e.g., such that the patient receives from about two to abouttwenty, or, for example, about six doses of the anti-CD79b antibody(e.g., anti-CD79b/CD3 TDB antibody). An initial higher loading dose,followed by one or more lower doses may be administered. The progress ofthis therapy is easily monitored by conventional techniques and assays.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-CD79b antibody.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1

Materials and Methods

A. Monoclonal Antibody Generation

Protein for immunization of mice was generated by transient transfectionof vectors that express Fc-tagged or His-tagged extra-cellular domain(ECD) of human CD79b into CHO cells. The proteins were purified from thetransfected cell supernatants on protein A columns and the identity ofthe protein confirmed by N-terminal sequencing. Ten Balb/c mice (CharlesRiver Laboratories, Hollister, Calif.) were hyperimmunized withrecombinant Fc-tagged or His-tagged ECD of human CD79b. B cells frommice demonstrating high antibody titers against the human CD79bimmunogen by direct ELISA, and specific binding to Ramos cells, werefused with mouse myeloma cells (X63.Ag8.653; American Type CultureCollection, Rockville, Md.) as previously described (Hongo, J. S. etal., Hybridoma, 14:253-260 (1995); Kohler, G. et al., Nature,256:495-497 (1975); Freund, Y. R. et al., J. Immunol., 129:2826-2830(1982)). After 10 to 12 days, the supernatants were harvested andscreened for antibody production and binding by direct ELISA and FACS asindicated above. Positive clones, showing the highest immunobindingafter the second round of subcloning by limiting dilution, were expandedand cultured for further characterization, including human CD79bspecificity and cross-reactivity. The supernatants harvested from eachhybridoma lineage were purified by affinity chromatography (Pharmaciafast protein liquid chromatography (FPLC); Pharmacia, Uppsala, Sweden)as previously described (Hongo, J. S. et al., Hybridoma, 14:253-260(1995); Kohler, G. et al., Nature, 256:495-497 (1975); Freund, Y. R. etal., J. Immunol., 129:2826-2830 (1982)). The purified antibodypreparations were then sterile filtered (0.2-Φm pore size; Nalgene,Rochester N.Y.) and stored at 4° C. in phosphate buffered saline (PBS).

B. Generation of TDBs

TDB antibodies were produced as full-length antibodies in theknob-into-hole format as human IgG1, as previously described (Atwell etal. J. Mol. Biol. 270: 26-35, 1997). Half antibodies were expressed ineither E. coli or Chinese hamster ovary (CHO) cells, purified by ProteinA-affinity chromatography, and the proper half antibody pairs wereannealed in vitro as described previously (Spiess et al. Nat.Biotechnol. 2013). If TDB antibody production was carried out in CHOcells, the antibody may include an aglycosylation mutation, for example,at residue N297 (e.g., N297G), such that the TDB antibody was aneffector-less variant and unable to initiate antibody-dependentcell-mediated cytotoxicity (ADCC).

After annealing, the anti-CD79b/CD3 TDB antibodies were purified byHydrophobic Interaction Chromatography (HIC) and characterized byanalytical gel filtration, mass spectrometry, and polyacrylamide gelelectrophoresis. The purified antibodies ran as a single peak (>99% ofthe signal) in gel filtration with less than 0.2% aggregates. Nohomodimers were detected by mass spectrometry.

C. Binding Affinity

Binding affinities for the each of the CD3/CD79b TDBs were tested byBiacore or FACS analysis. Briefly, for Biacore binding assays, humanCD3γε was immobilized on Biacore Series S CM5 sensor chip using theamine coupling kit from Biacore and anti-CD79b/CD3 TDB antibodies or Fabvariants thereof were in the flow through. For FACS binding assays, BJABcells (for B cell antigens) or other cell lines as specified wereincubated with various concentrations of TDB antibodies at 4° C. for 30minutes, then cells were washed and incubated with 2^(nd) antibody(anti-huIgG-PE; BD Bioscience) for another 15 minutes, before cells werewashed again and ready for FACS analysis.

D. In Vitro B Cell Killing and T Cell Activation Assays

The generated anti-CD79b/CD3 TDB antibodies were tested for theirability to support B cell killing and the activation of cytotoxic Tcells. In these assays, PBMCs were isolated from whole blood of healthydonors by Ficoll separation. Briefly, human blood was collected inheparinized syringes, and PBMC were isolated using Leucosep (GreinerBio-one, cat #227290P) and Ficoll Paque Plus (GE Healthcare Biosciences,cat #95038-168), as recommended by the manufacture. If needed CD4+T andCD8+ T cells were separated with Miltenyi kits according tomanufacturer's instructions.

Cells were washed in RPMI medium containing 10% FBS, supplemented withGlutaMax (Gibco, cat #35050-061), penicillin & streptomycin (Gibco, cat#15140-122), and ˜0.2 million suspended cells were added to a 96-wellU-bottom plate. Cells were cultured in RPMI1640 supplemented with 10%FBS (Sigma-Aldrich) at 37° C. in a humidified standard cell cultureincubator. For BJAB cell killing assays, 20,000 BJAB cells wereincubated with effector cells either as huPBMCs or purified T cells asindicated ratios per assay, in the presence of various concentrations ofTDB antibodies for 24 hours, unless otherwise specified. For endogenousB cell killing assays, 200,000 huPBMCs were incubated with variousconcentrations of anti-CD79b/CD3 TDB antibodies for 24 hours, unlessotherwise specified.

After culturing, cells were washed with FACS buffer (0.5% BSA, 0.05% NaAzide in PBS). Cells were then stained in FACS buffer, washed with FACSbuffer and suspend in 100 ul of FACS buffer containing 1 ug/ml PropidiumIodide. Data was collected on a FACSCalibur flow cytometer and analyzedusing FlowJo. Live B cells were gated out as PI-CD19+ or PI-CD20+ Bcells by FACS, and absolute cell count was obtained with FITC beadsadded to reaction mix as internal counting control. % of cell killingwas calculated based on non-TDB treated controls. Activated T cells weredetected by CD69 and CD25 surface expression using anti-CD69-FITC (BD,cat #555530) and anti-CD25-PE (BD, cat #555432).

D. In Vivo Efficacy

50 SCID.bg mice were inoculated with 5 million BJAB-lucanti-CD79b-MC-vc-PAB-MMAE resistant model T1.1 X1 cells in HBSSsubcutaneously in a volume of 0.2 mL per mouse in the rightunilateral-thoracic (not to exceed 200 ul) or a mixture of 5 millionBJAB-luc anti-CD79b-MC-vc-PAB-MMAE resistant model T1.1 X1 cells and 10million PBMCs in HBSS a volume of 0.2 ml (not to exceed 200 ul). Thiswas a preventative study so inoculation and treatment were administeredon Day 0.

There were five study groups: 1) 5 million BJAB-lucanti-CD79b-MC-vc-PAB-MMAE resistant model T1.1 X1, Vehicle, qwx2, IV; 2)5 million BJAB-luc anti-CD79b-MC-vc-PAB-MMAE resistant model T1.1 X1,0.5 mg/kg anti-CD79 TDB, qwx2, IV; 3) 5 million BJAB-lucanti-CD79b-MC-vc-PAB-MMAE resistant model T1.1 X1+10×10{circumflex over( )}6 PBMCs (pre-mixed), Vehicle, qwx2, IV; 4) 5 million BJAB-lucanti-CD79b-MC-vc-PAB-MMAE resistant model T1.1 X1+10×10{circumflex over( )}6 PBMCs (pre-mixed), 0.5 mg/kg anti-CD79 TDB (CD79b.A7.v14b/38E4v1),qwx2, IV; and 5) 5 BJAB-luc anti-CD79b-MC-vc-PAB-MMAE resistant modelT1.1 X1, 8 mg/kg BJAB-luc anti-CD79b-MC-vc-PAB-MMAE, once, IV. PBMCswere from Buffy Coat Donor, cultured overnight in non-activatingcondition, inoculated as a mixture with the BJAB cells. All treatmentswere administered i.v., tail vein, volume=0.1 ml (not to exceed 200 ul).Tumors were measured 1-2 times per week. Body weights were measured1-2×/week up to 14 days after the final treatment.

1. Selection of CD79b TDB-Anti-CD79b Antigen Arm

Antibody—drug conjugates (ADC) have been generated (such as thehumanized anti-CD79b antibody (humanized SN8) conjugated tomonomethylauristatin E (MMAE) by a protease cleavable linker), which hasshown in the clinic to be efficacious for the treatment of NHL. See U.S.Pat. No. 8,088,378 and Morschhauser et al., “4457 Updated Results of aPhase II Randomized Study (ROMULUS) of Polatuzumab Vedotin orPinatuzumab Vedotin Plus Rituximab in Patients with Relapsed/RefractoryNon-Hodgkin Lymphoma” 56^(th) ASH Annual Meeting and Exposition: Dec.6-9, 2014.

Based on the clinical success of the anti-CD79b ADC, the humanized SN8antibody was in a T-cell dependent bispecific (TDB) antibody format toharness the high cytotoxic potential of T cells in eradicating tumorcells. See U.S. Pat. No. 8,088,378, which is hereby incorporated byreference in its entirety. An anti-CD3 (e.g., UCHT1.v9; see, e.g., Zhuet al. Int. J. Cancer 62:319-324 (1995))/anti-CD79b (e.g., SN8.v28)bispecific knob & hole (K&H) was generated as described above. However,in endogenous B cell killing assaying using two different donors asdescribed above, poor B cell killing activity for the UCHT1.v9/SN8.v28bispecific K&H was observed: the EC₅₀ was 357 ng/mL and 120 ng/mL thecell killing assay.

A second anti-CD3 (e.g., UCHT1.v9)/anti-CD79b TDB was generated using2F2 as the anti-CD79b antibody arm. 2F2 had shown in vitro promise as ananti-CD79b ADC. See e.g., US20090068202, incorporated by reference inits entirety. In addition, the CD79b arm antibody SN8.v28 was modifiedin an attempt to improve cell killing (SN8.new (VH SEQ ID NO:37 and VLSEQ ID NO:38)). As shown in FIG. 1A (endogenous B cell killing assay)and FIG. 1B (BJAB cell killing assay), SN8.v28/UCHT1.v9 bispecific K&Hantibodies, SN8.new/UCHT1.v9 bispecific K&H antibodies, as well as2F2/UCHT1.v9 bispecific K&H antibodies resulted in poor B cell killingactivity.

Monoclonal anti-CD79b antibodies were generated as described above. Twoof these anti-CD79b antibodies (CD79b.F6 and CD79b.A7) were also testedas bispecific bisfab format anti-CD79b/CD3 antibodies. As shown in FIG.1C, in an endogenous B cell killing assay, CD79b.F6/UCHT1.v9 bispecificbisfab displayed improved B cell killing compared to SN8.v28/UCHT1.v9bispecific K&H (EC₅₀ of 33 ng/mL compared to 189 ng/mL). Further, asshown in FIG. 1C, in the endogenous B cell killing assay, the bispecificbisfab CD79b.A7/UHT1.v9 dramatically improved B cell killing compared toeither bispecific bisfab CD79b.F6/UCHT1.v9 or SN8.v28/UCHT1.v9bispecific K&H (EC₅₀ 12 ng/mL compared to 33 ng/mL and 189 ng/mL). Thebispecific bisfab CD79b.A7/UHT1.v9 was further tested for endogenous Bcell killing and CD8+ T cell activation using additional donors. Asshown in FIGS. 2A and C, in the endogenous B cell killing assay asdescribed above, the bispecific bisfab CD79b.A7/UHT1.v9 using twodifferent donors resulted in efficient B cell killing with an EC₅₀ of7.0 ng/mL and 18 ng/mL, respectively. At the same time, as shown inFIGS. 2B and D in the CD8+ T cell activation assay as described above,the bispecific bisfab CD79b.A7/UHT1.v9 resulted in efficient activationof T cells as evidenced by % of CD69+CD25+ T cells in CD8+ T cells withan EC₅₀ of 17 ng/mL and 17 ng/mL, respectively.

In order to better understand the difference in the B cell killing and Tcell activation of the different anti-CD79b antigen arms in theanti-CD79b/CD3 TDB antibodies, the properties of the differentanti-CD79b antigen arms were analyzed. Binding of CD79b.A7 to BJAB cellsin a FACS assay were competed by a 21-amino acid peptideARSEDRYRNPKGSACSRIWQS (SEQ ID NO:63) that corresponds to the NH₂ terminiof huCD79b, but not the 21-amino acid peptide AKSEDLYPNPKGSACSRIWQS (SEQID NO:64) that correspond to the NH₂ termini of cynoCD79b (data notshown). This is similar to the results of 2F2 and SN8 as described inZheng et al. Mol. Cancer Ther. 8(10):2937-2947 (2009). Therefore, theepitope on CD79b does not appear to account for the difference in B cellkilling and T cell activation between SN8.v28/UCHT1.v9, 2F2/UCHT1.v9,and CD79.A7.

The monovalent and bivalent anti-CD79b antibody binding affinity wasalso evaluated in order to better understand the contribution, if any,to B cell killing activity. Binding affinity of dual arm, bivalentanti-CD79b antibodies and bispecific anti-CD79b/CD3 antibodies usingBJAB cells were also analyzed. Initial experiments indicated that thebinding affinity by EC₅₀ on BJAB cell of the dual arm, bivalentanti-CD79b antibody SN8.v28 was 0.04 μg/mL, while the binding affinityof by EC₅₀ on BJAB cell of the anti-CD79b/CD3 bispecific K&H(SN8.v28/UCHT1.v9) was only 3.7 μg/mL. Similarly as shown in FIG. 3A,the binding affinity by EC₅₀ on BJAB cell of the dual arm, bivalentanti-CD79b antibody 2F2 was significantly higher than the anti-CD79b/CD3bispecific K&H (SN8.v28/UCHT1.v9, SN8new/UCHT1.v9, and 2F2/UCHT.v9). Thebinding affinity of the additional anti-CD79 antibodies, CD79b.F6 andCD79b.A7, were tested in the dual arm, bivalent anti-CD79b antibodyformat as well as the bisfab and K&H bispecific anti-CD79b/CD3antibodies. As shown in FIG. 3B, the binding affinity of dual arm,bivalent anti-CD79b antibody CD79.A7 by EC₅₀ on BJAB cell was 0.3 μg/mL.The binding affinity by EC₅₀ on BJAB cell of the bispecific bisfabanti-CD79b/CD3 (CD79b.A7/UCHT1.v9) was lower than the dual arm, bivalentanti-CD79b antibody CD79.A7, but still relatively high 1.4 μg/mL. Thebinding affinity by EC₅₀ on BJAB cell of the dual arm, bivalentanti-CD79b antibody CD79b.F6 and the K&H and bisfab bispecificanti-CD79b/CD3 antibodies (SN8.v28/UCHT1.v9 and CD79b.F6/CHT1.v9,respectively) was significantly lower. Based on this data, themonovalent binding affinity correlated with the extent of endogenous Bcell depletion and % B cell killing.

2. Humanization of Anti-CD79b Antigen Arm

Monoclonal antibody CD79b.A7 was humanized as described above. Residuenumbers are according to Kabat et al., Sequences of proteins ofimmunological interest, 5th Ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).

Variants constructed during the humanization of CD79b.A7 were assessedin the form of an IgG. The VL and VH domains from murine CD79b.A7 werealigned with the human VL kappa II (VLK2) and human VH subgroup I (VHI)consensus sequences. Hypervariable regions from the murine antibodieswere engineered into VLK2 and VHI acceptor frameworks. Specifically,from the muCD79b.A7 domain, positions 24-34 (L1), 50-56 (L2) and 89-97(L3) were grafted into VLK2 and from the muCD79b.A7 VH domain, positions26-35 (H1), 50-65 (H2) and 93-102 (H3) were grafted into VHI.

The binding affinity of the antibodies in this section was determined byBIAcore™ T100. Briefly, BIAcore™ research grade CM5 chips were activatedwith 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) andN-hydroxysuccinimide (NHS) reagents according to the supplier'sinstructions. Human CD79a fused with an Fc domain at the C-terminus wascoupled to the chips. To achieve more monovalent binding events, lowdensity of ˜12 response units (RU) was immobilized in each flow cell.For measuring the apparent affinity of the antibodies, ˜370 RU of theantigen was immobilized. Unreacted coupling groups were blocked with 1Methanolamine. For kinetics measurements, three-fold serial dilutions ofantibody was injected in PBS-T buffer (0.05% surfactant P20 in PBS) at25° C. with a flow rate of 30 μl/min. 10 mM glycine, pH 1.7 was used asregeneration buffer at 30 ul/min flow rate for 1 minute. Associationrates (k_(on)) and dissociation rates (k_(off)) were calculated using a1:1 Langmuir binding model (BIAcore™ T100 Evaluation Software version2.0). The equilibrium dissociation constant (Kd) was calculated as theratio k_(off)/k_(on).

The humanized CDR graft of CD79b.A7 (CD79b.A7.v1) did not bind to CD79b.Thus additional humanized variants were made to evaluate thecontribution of mouse framework vernier positions towards binding. Sixadditional light chains (VL1: CDRs graft+(Y36L), VL2: CDRsgraft+(Y36L+L46C), VL3: CDRs graft+(I2V+Y36L+L46C), VL4: CDRsgraft+(Y36L+L46S), VL5: CDRs graft+(I2V+Y36L+L46S), VL6: CDRsgraft+(L46S)) and seven additional heavy chains (VH1a: CDRsgraft+(A93S), VH1b: CDRs graft+(R71V+A93S), VH1c: CDRsgraft+(V67A+I69L+R71V+A93S), VH1d:CDRs graft+(V67A+I69L+R71V+T73K+A93S),VH1e: CDRs graft+(V67A+R71V+A93S), VH1f: CDRs graft+(I69L+R71V+A93S),and VH1g: CDRs graft+(V67A+I69L)) were constructed and combined togenerate the variants in Table 2. Based on the affinities of thesevariants, Y36L and L46C in the light chain appear to be key mousevernier residues. Surprisingly, when position 46 was changed to a serinein order to avoid the use of a free cysteine, the affinity of variantswith this change improved dramatically. In the heavy chain, V67A, I69L,R71V and A93S also contributed to CD79b binding; however R71V appearedto be the key mouse vernier residues based on this mutational analysis.The affinities in Table 2 are all apparent affinities based on bivalentIgG binding to CD79b immobilized at a low density to approximatemonovalent binding. See Table 2 below.

TABLE 2 CD79b.A7 K2 nM graft VL1 VL2 VL3 VL4 VL5 VL6 VH1 graft v1 v2 v3No NDB  320 nM Bind- ing VH1a v4 v5 v6 NDB NDB 3800 nM VH1b v7 v8 v9 v27NDB NDB 7050 nM 8 nM VH1c v10 v11 v12 v13 v14 v15 v26 NDB NDB  20 nM 716nM 5 nM 5 nM 23 nM VH1d v16 v17 v18 v19  117 nM  88 nM 4 nM 4 nM VH1ev20 v23 8 nM 20 nM VH1f v21 v24 8 nM 39 nM VH1g v22 v25 16 nM  68 nM

Binding affinity was further tested by FACS analysis, BJAB cells wereincubated with various anti-CD79b antibodies for 30 minutes on ice. Atthe end of the incubation, cells were washed with ice cold FACS buffer(1×PBS, 2% BSA, 2 mM EDTA), followed by incubation with PE-labeled mouseanti-human IgG antibody (BD bioscience #555787). Flow cytometry analysiswas done on a BD LSR analyzer. Bivalent binding was expressed as MeanFluorescence Intensity (MFI) of PE fluorophore. The binding ofchCD79b.A7, huCD79b.A7.v12 and huCD79b.A7.v14 to BJAB-luciferase cellswas at an EC₅₀ of 124 ng/mL, 400 ng/mL, and 68 ng/mL, respectively.

Binding affinity of the humanized CD79.A7.v14 of monovalent and bivalentwas tested as described above. As shown in FIG. 3C, the monovalentCD79.A7.v14 (in the K&H TDB format CD79.A7.v14/40G5c) had an EC₅₀ of 220ng/ml while the bivalent, dual arm CD79A7.v14 had an EC₅₀ of 46.8 ng/ml.

The humanized antibody CD79.A7.v14 was tested under thermal stress (40°C., pH 5.5, 2 weeks) and 2,2′-azobis (2-amidinopropane) hydrochloride(AAPH) Analysis. Samples were thermally stressed to mimic stability overthe shelf life of the product. Samples were buffer exchanged into 20 mMHis Acetate, 240 mM sucrose, pH 5.5 and diluted to a concentration of 1mg/mL. One mL of sample was stressed at 40 C for 2 weeks and a secondwas stored at −70 C as a control. Both samples were then digested usingtrypsin to create peptides that could be analyzed using liquidchromatography (LC)-mass spectrometry(MS) analysis. For each peptide inthe sample retention time, from the LC as well as high resolutionaccurate mass and peptide ion fragmentation information (amino acidsequence information) were acquired in the MS. Extracted ionchromatograms (XIC) were taken for peptides of interest (native andmodified peptide ions) from the data sets at a window of +−10 ppm andpeaks were integrated to determine area. Relative percentages ofmodification were calculated for each sample by taking the (area of themodified peptide) divided by (area of the modified peptide plus the areaof the native peptide) multiplied by 100.

W33 in CDR-H1 and M62 in CDR-H2 of CD79b.A7.v14 were shown to besusceptible to oxidation (W33 oxidation increased by 73.7% and M62oxidation increased by 64.8%). Variants of CD79b.A7.v14 antibodies weretested to determine if potential oxidation could be reduced withoutaffecting binding to huCD79b. The variant, CD79b.A7.v14b, eliminatedthese potential oxidation problems by changing these regions to matchthe human VH1 consensus (W33Y, M62K, K64Q and D65G). These changes didnot alter the affinity for CD79b binding. See data not shown.

3. Selection of CD79b TDB-Anti-CD3 Antigen Arm

The effect of the CD3 binding domain pairing on efficiency of anti-CD79bTDB antibody B cell killing was analyzed. The anti-CD79b.A7.v14 antibodywas tested in combination with different anti-CD3 antibody bindingdomains including 40G5c and 38E4v1. 200,000 PBMCs were incubated with orwithout anti-CD79b/CD3 TDB antibody for 48 hours. The percent of B cellkilling in FIG. 5A was calculated as follows: (live B cell numberwithout TDB—live B cell number with TDB)/(live B cell number withoutTDB)*100. T cell activation as shown in FIG. 5B was measured by gatingon CD69⁺/CD25⁺ cells in CD8⁺ T cell population. As shown in FIGS. 5A andB, K&H CD79b.A7.v14/40G5c showed poor CD8+T activation and lowpercentage of endogenous B cell killing. In additional experiments (datanot shown), the anti-CD79b/CD3 TDB antibody (CD79b.A7.v14/40G5c K&H)showed an EC₅₀ of 1.1 and 2.3 ng/mL for CD8+T activation and an EC₅₀ of2658 and 288 ng/mL for endogenous B cell killing using two differentdonors. In contrast, as shown in FIGS. 5A and B, the anti-CD79b/CD3 TDBantibody (CD79b.A7.v14/38E4v1 K&H) showed substantially improved CD8+ Tcell activation and percentage of endogenous B cell killing—an EC₅₀ of15 ng/mL for endogenous B cell killing compared to CD79b.A7.v14/40G5cK&H. In additional experiments (data not shown), the anti-CD79b/CD3 TDBantibody (CD79b.A7.v14/40G5c K&H) showed an EC₅₀ of 401, 14, 1.5, 10,12, and 16 ng/mL for endogenous B cell killing using six differentdonors.

B cell killing and T cell activation activity of the anti-CD79b/CD3 TDBantibody (CD79b.A7.v14/40G5c K&H) were also tested in BJAB and WSU-DLCL2cell lines as described above and in the FIG. 6 Figure Legend. As shownin FIGS. 6A and B, the anti-CD79b/CD3 TDB antibody (CD79b.A7.v14/40G5cK&H) showed significant CD8+T activation and percentage of B cellkilling—an EC₅₀ of 85 ng/mL for BJAB B cell killing and 82 ng/mL forWSU-DLCL2 B cell killing. The EC₅₀ for CD8+ T cell activation with BJABand WSU-DLCL2 B cells was 18 and 39 ng/mL, respectively.

B cell killing activity of the anti-CD79b/CD3 TDB antibody(CD79b.A7.v14b/40G5c K&H) was tested in various cell lines as describedin FIG. 7A-B. The HT cell line is a non-CD79b expressing cell. Thepercent of B cell killing was calculated as follows: (live B cell numberwithout TDB—live B cell number with TDB)/(live B cell number withoutTDB)*100. As shown in FIG. 7A, in a dose response curve of B cellkilling for BJAB, WSU-DLCL2, and OCI-LY-19 cells, the EC₅₀ of 8.87,2.63, and 17.41 ng/mL for OCI-Ly-19, BJAB, and WSU-DLCL2 B cell killing,respectively. FIG. 7B shows significant B cell killing with 5000 ng/mlanti-CD79/CD3 TDB antibody (CD79b.A7.v14b/38E4v1 K&H) across multiplecell lines (duplicate, average±STD).

The varied efficacies of the generated TDB antibodies with bispecificfor CD3 and CD79b, underscore the critical and unpredictablecontributions of both antibody arms in the generation of an exemplaryTDB possessing high efficacy

4. In Vitro and In Vivo Efficacy in Anti-CD79b-MC-Vc-PAB-MMAE ResistantB Cells

The B cell killing activity of anti-CD79b/CD3 TDB antibodies(CD79b.A7.v14b/38E4v1) in vitro and in vivo were also tested inanti-CD79b-MC-vc-PAB-MMAE Resistant B cells. BJAB cell variants(BJAB-CD79b ADC-R T1.1 and BJAB-SN8v28vcE CD79b ADC-R T1.2) were derivedfrom non-responsive BJAB xenograft tumors in anti-CD79b-MC-vc-PAB-MMAEResistant B cells treated mice. As shown in FIG. 8A, the anti-CD79b/CD3TDB (CD79b.A7.v14b/38E4v1) was very effective in BJAB as well asanti-CD79b-MC-vc-PAB-MMAE Resistant BJAB cells cell killing in vitro.Further, as shown in FIG. 8B, anti-CD79b/CD3 TDB antibody(CD79b.A7.v14b/38E4v1) prevents anti-CD79b-MC-vc-PAB-MMAE Resistant BJABtumor growth in vivo.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

NAME SEQUENCE SEQ ID NO Human CD79bRFIARKRGFT VKMHCYMNSA SGNVSWLWKQ EMDENPQQLK  1 precursor; Acc.LEKGRMEESQ NESLATLTIQ GIRFEDNGIY FCQQKCNNTS No. NP_000617.1;EVYQGCGTEL RVMGFSTLAQ LKQRNTLKDG IIMIQTLLII signal sequence =LFIIVPIFLL LDKDDSKAGM EEDHTYEGLD IDQTATYEDI amino acids 1 toVTLRTGEVKW SVGEHPGQE 28 Human mature AR SEDRYRNPKG SACSRIWQSP RFIARKRGFT 2 CD79b, without VKMHCYMNSA SGNVSWLWKQ EMDENPQQLK LEKGRMEESQsignal sequence; NESLATLTIQ GIRFEDNGIY FCQQKCNNTS EVYQGCGTELamino acids 29 to RVMGFSTLAQ LKQRNTLKDG IIMIQTLLII LFIIVPIFLL 229LDKDDSKAGM EEDHTYEGLD IDQTATYEDI VTLRTGEVKW SVGEHPGQE CD79b.A7, TYWMN  3CD79b.A7.v14 CD79b.A7.v15 CD79b.A7.v18 CD79b.A7.v19 CD79b.A7.v20CD79b.A7.v21 HVR-H1 CD79b.A7.v14b TYYMN  4 HVR-H1 ConsensusTYX₁MN, wherein X₁ is W or Y  5 HVR-H1 CD79b.A7, MIDPSDSETHYNQMFKD  6CD79b.A7.v14 CD79b.A7.v15 CD79b.A7.v18 CD79b.A7.v19 CD79b.A7.v20CD79b.A7.v21 HVR-H2 CD79b.A7.v14b MIDPSDSETHYNQKFQG  7 HVR-H2 ConsensusMIDPSDSETHYNQX₂FX₃X₄, wherein X₂ is M or K, X₃ is  8 HVR-H2K or Q, and X₄ is D or G. CD79b.A7, SLAF  9 CD79b.A7.v14 CD79b.A7.v14bCD79b.A7.v15 CD79b.A7.v18 CD79b.A7.v19 CD79b.A7.v20 CD79b.A7.v21 HVR-H3CD79b.A7, KSSQSLLDSDGKTYLN 10 CD79b.A7.v14 CD79b.A7.v14b CD79b.A7.v15CD79b.A7.v18 CD79b.A7.v19 CD79b.A7.v20 CD79b.A7.v21 HVR-L1 CD79b.A7,LVSKLDS 11 CD79b.A7.v14 CD79b.A7.v14b CD79b.A7.v15 CD79b.A7.v18CD79b.A7.v19 CD79b.A7.v20 CD79b.A7.v21 HVR-L2 CD79b.A7, WQGTHFPQT 12CD79b.A7.v14 CD79b.A7.v14b CD79b.A7.v15 CD79b.A7.v18 CD79b.A7.v19CD79b.A7.v20 CD79b.A7.v21 HVR-L3 K2H1EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEW 13 Heavy ChainIGWINPGSGNTNYAQKFQGRVTITRDTSTSTAYLELSSLRSEDTAVY Variable RegionYYCARFDYWGQGTLVTVSS (V_(H)) K2H1DIVMTQTPLSLPVTPGQPASISCRSSQSLLHSSGNTYLDWYLQKPGQ 14 Light ChainSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQ VariableQAIQFPFTFGQGTKVEIK Region(V_(L)) CD79b.A7 V_(H)QVQLQQPGVELVRPGASVKLSCKASGYTFTTYWMNWVRQRPGQGLDW 15IGMIDPSDSETHYNQMFKDKATLTVDKSSSTAYIQLNSLTSEDSAVY YCSRSLAFWGQGTLVTVSACD79b.A7 V_(L) DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQ 16SPKCLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCW QGTHFPQTFGGGTKLEIKCD79b.A7.v14 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMNWVRQAPGQGLEW 17IGMIDPSDSETHYNQMFKDRATLTVDTSTSTAYLELSSLRSEDTAVY YCSRSLAFWGQGTLVTVSSCD79b.A7.v14 V_(L) DIVMTQTPLSLPVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQ 18SPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCW QGTHFPQTFGQGTKVEIKCD79b.A7.v14b V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMNWVRQAPGQGLEW 19IGMIDPSDSETHYNQKFQGRATLTVDTSTSTAYLELSSLRSEDTAVY YCSRSLAFWGQGTLVTVSSCD79b.A7.v14b V_(L) DIVMTQTPLSLPVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQ 20SPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCW QGTHFPQTFGQGTKVEIKCD79b.A7.v15 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMNWVRQAPGQGLEW 21IGMIDPSDSETHYNQMFKDRATLTVDTSTSTAYLELSSLRSEDTAVY YCSRSLAFWGQGTLVTVSSCD79b.A7.v15 V_(L) DVVMTQTPLSLPVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQ 22SPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCW QGTHFPQTFGQGTKVEIKCD79b.A7.v18 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMNWVRQAPGQGLEW 23IGMIDPSDSETHYNQMFKDRATLTVDKSTSTAYLELSSLRSEDTAVY YCSRSLAFWGQGTLVTVSSCD79b.A7.v18 V_(L) DIVMTQTPLSLPVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQ 24SPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCW QGTHFPQTFGQGTKVEIKCD79b.A7.v19 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMNWVRQAPGQGLEW 25IGMIDPSDSETHYNQMFKDRATLTVDKSTSTAYLELSSLRSEDTAVY YCSRSLAFWGQGTLVTVSSCD79b.A7.v19 V_(L) DVVMTQTPLSLPVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQ 26SPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCW QGTHFPQTFGQGTKVEIKCD79b.A7.v20 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMNWVRQAPGQGLEW 27IGMIDPSDSETHYNQMFKDRATITVDTSTSTAYLELSSLRSEDTAVY YCSRSLAFWGQGTLVTVSSCD79b.A7.v20 V_(L) DIVMTQTPLSLPVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQ 28SPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCW QGTHFPQTFGQGTKVEIKCD79b.A7.v21 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMNWVRQAPGQGLEW 29IGMIDPSDSETHYNQMFKDRVTLTVDTSTSTAYLELSSLRSEDTAVY YCSRSLAFWGQGTLVTVSSCD79b.A7.v21 V_(L) DIVMTQTPLSLPVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQ 30SPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCW QGTHFPQTFGQGTKVEIKSN8.new HVR-H1 SYWIE 31 SN8.new HVR-H2 EILPGGGDTNYNEIFKG 32SN8.new HVR-H3 RVPIRLDY 33 SN8.new HVR-L1 KASQSVDYDGDSFLN 34SN8.new HVR-L2 AARKLGR 35 SN8.new HVR-L3 QQSNEDPLT 36 SN8.new V_(H)EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEW 37VGEILPGGGDTNYNEIFKGRFTISADTSKNTAYLQMNSLRAEDTAVY YCTRRVPIRLDYWGQGTLVTVSSSN8.new V_(L) DIQMTQSPSSLSASVGDRVTITCKASQSVDYDGDSFLNWYQQKPGKA 38PKLLIYAARKLGRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ SNEDPLTFGQGTKVEIK40G5c HVR-H1 NYYIH 39 40G5c HVR-H2 WIYPGDGNTKYNEKFKG 40 40G5c HVR-H3DSYSNYYFDY 41 40G5c HVR-L1 KSSQSLLNSRTRKNYLA 42 40G5c HVR-L2 WASTRES 4340G5c HVR-L3 TQSFILRT 44 38E4v1 HVR-H1 SYYIH 45 38E4v1 HVR-H2WIYPENDNTKYNEKFKD 46 38E4v1 HVR-H3 DGYSRYYFDY 47 38E4v1 HVR-L1KSSQSLLNSRTRKNYLA 48 38E4v1 HVR-L2 WTSTRKS 49 38E4v1 HVR-L3 KQSFILRT 50UCHT1 v9 HVR-H1 GYTMN 51 UCHT1 v9 HVR-H2 LINPYKGVSTYNQKFKD 52UCHT1 v9 HVR-H3 SGYYGDSDWYFDV 53 UCHT1 v9 HVR-L1 RASQDIRNYLN 54UCHT1 v9 HVR-L2 YTSRLES 55 UCHT1 v9 HVR-L3 QQGNTLPWT 56 40G5c V_(H)EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEW 57IGWIYPGDGNTKYNEKFKGRATLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSS 40G5c V_(L)DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPG 58QPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC TQSFILRTFGQGTKVEIK38E4v1 V_(H) EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQGLEW 59IGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSS 38E4v1 V_(L)DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPG 60QSPKLLIYWTSTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC KQSFILRTFGQGTKVEIKUCHT1v9 V_(H) EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKDLEW 61VALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS UCHT1v9 V_(L)DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLL 62IYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTL PWTFGQGTKLELK Peptide 1ARSEDRYRNPKGSACSRIWQS 63 Peptide 2 AKSEDLYPNPKGSACSRIWQS 64

What is claimed is:
 1. A bispecific antibody that binds to CD79b andCD3, wherein the bispecific antibody comprises: (a) a CD79b bindingdomain comprising the following six hypervariable regions (HVRs): anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 5, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 8, an HVR-H3 comprisingthe amino acid sequence of SEQ ID NO: 9, an HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 10, an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 11, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO, 12; and (b) a CD3 binding domain.
 2. Thebispecific antibody of claim 1, wherein CD3 binding domain comprises thefollowing six HVRs: an HVR-H1 comprising the amino acid sequence of SEQID NO:45, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:46,an HVR-H3 comprising the amino acid sequence of SEQ ID NO:47, an HVR-L1comprising the amino acid sequence of SEQ ID NO:48, an HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:49, and an HVR-L3 comprising theamino acid sequence of SEQ ID NO:50.
 3. The bispecific antibody of claim2, wherein the CD3 binding domain comprises (a) a VH sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:59;(b) a VL sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:60; or (c) a VH sequence as in (a) and a VLsequence as in (b).
 4. The bispecific antibody of claim 1, wherein theCD3 binding domain comprises a VH sequence of SEQ ID NO:59 and a VLsequence of SEQ ID NO:60.
 5. A bispecific antibody that binds to CD79band CD3, wherein the bispecific antibody comprises: (a) a CD79b bindingdomain comprising the following six hypervariable regions (HVRs): anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 3, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 6, an HVR-H3 comprisingthe amino acid sequence of SEQ ID NO: 9, an HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 10, an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 11, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO, 12; and (b) a CD3 binding domain comprising thefollowing six HVRs: an HVR-H1 comprising the amino acid sequence of SEQID NO:45, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:46,an HVR-H3 comprising the amino acid sequence of SEQ ID NO:47, an HVR-L1comprising the amino acid sequence of SEQ ID NO:48, an HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:49, and an HVR-L3 comprising theamino acid sequence of SEQ ID NO:50.
 6. The bispecific antibody of claim5, wherein the CD3 binding domain comprises (a) a VH sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:59;(b) a VL sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:60; or (c) a VH sequence as in (a) and a VLsequence as in (b).
 7. The bispecific antibody of claim 5, wherein theCD3 binding domain comprises a VH sequence of SEQ ID NO:59 and a VLsequence of SEQ ID NO:60.
 8. A bispecific antibody that binds to CD79band CD3, wherein the bispecific antibody comprises: (a) a CD79b bindingdomain comprising the following six hypervariable regions (HVRs): anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 4, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 7, an HVR-H3 comprisingthe amino acid sequence of SEQ ID NO: 9, an HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 10, an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 11, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO, 12; and (b) a CD3 binding domain comprising thefollowing six HVRs: an HVR-H1 comprising the amino acid sequence of SEQID NO:45, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:46,an HVR-H3 comprising the amino acid sequence of SEQ ID NO:47, an HVR-L1comprising the amino acid sequence of SEQ ID NO:48, an HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:49, and an HVR-L3 comprising theamino acid sequence of SEQ ID NO:50.
 9. The bispecific antibody of claim8, wherein the CD3 binding domain comprises (a) a VH sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:59;(b) a VL sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:60; or (c) a VH sequence as in (a) and a VLsequence as in (b).
 10. The bispecific antibody of claim 8, wherein theCD3 binding domain comprises a VH sequence of SEQ ID NO:59 and a VLsequence of SEQ ID NO:60.
 11. The bispecific antibody of claim 8,wherein the CD79b binding domain comprises (a) a VH sequence having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 19;(b) a VL sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:20; or (c) a VH sequence as in (a) and a VLsequence as in (b).
 12. The bispecific antibody of claim 8, wherein theCD79b binding domain comprises a VH sequence of SEQ ID NO: 19 and a VLsequence of SEQ ID NO:20.
 13. A bispecific antibody that binds to CD79band CD3, wherein the bispecific antibody comprises: (a) a CD79b bindingdomain comprising a VH sequence of SEQ ID NO: 19 and a VL sequence ofSEQ ID NO:20, and (b) a CD3 binding domain comprising a VH sequence ofSEQ ID NO:59 and a VL sequence of SEQ ID NO:60.
 14. The bispecificantibody of claim 8, wherein the bispecific antibody has a B cellkilling EC50 of less than about 100 ng/mL.
 15. The bispecific antibodyof claim 14, wherein the B cell killing is endogenous B cell killing.16. The bispecific antibody of claim 8, wherein the bispecific antibodyhas a cytotoxic T cell activation EC50 is less than about any of 50ng/mL.
 17. The bispecific antibody of claim 1, wherein (a) the CD3binding domain comprises a Fc domain, wherein the Fc domain comprisesT366S, L368A, Y407V, and N297G substitution mutations according EUnumbering and (b) the CD79b binding domain comprises a Fc domain,wherein the Fc domain comprises T366W and N297G substitution mutationsaccording EU numbering.
 18. An isolated nucleic acid encoding thebispecific antibody of claim
 1. 19. A vector comprising the isolatednucleic acid of claim
 18. 20. A host cell comprising the vector of claim19.
 21. A method of producing a bispecific antibody, the methodcomprising culturing the host cell of claim 20 in a culture medium. 22.A pharmaceutical composition comprising the bispecific antibody ofclaim
 1. 23. The bispecific antibody of claim 15, wherein the B cellkilling is cell line B cell killing using a cell line selected from thegroup consisting of BJAB cell line, WSU-CLCL2 cell line, and OCI-Ly-19cell line.